Method for treating subjects suffering from chronic ulcers

ABSTRACT

A method, material, and kit for promoting neutrophils and monocytes to localize at a chronic ulcer site, promoting formation of a multi-layered cell structure in the ulcer site, promoting conversion of monocytes to macrophages, promoting secretion of the patient&#39;s own growth factors, promoting tissue proliferation and cell migration, promoting production and cross-linking of collagen at the chronic ulcer site, promoting growth of endothelial cells, promoting angiogenesis that was stalled at the chronic ulcer site, promoting formation of a vascular network and granulation, promoting oxygenation of the chronic ulcer site, and reducing one or more of purulent drainage, erythema, pain, warming, tenderness, induration, and bleeding at the chronic ulcer site.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 16/845,633, filedApr. 10, 2020, which claims priority benefit of U.S. provisionalapplication No. 62/832,417, filed Apr. 11, 2019, the disclosures ofwhich are incorporated herein by reference in their entireties.

FIELD OF THE DISCLOSURE

This disclosure relates to methods and materials for treating subjectssuffering from chronic ulcers, in particular chronic skin ulcers.

BACKGROUND

Chronic ulcers are persistent, non-healing or slow-healing ulcersaffecting millions of persons each year, particularly the elderly anddiabetics. Wounds that fail to progress through the healing process in atimely manner, e.g. 4-5 weeks, are often referred to as chronic ulcers.Such ulcers may last months or years, recur in a majority of patients,can lead to loss of function and decreased quality of life, and are asignificant cause of morbidity. Some common features shared by each ofthese ulcers include prolonged or excessive inflammation, persistentinfections, formation of drug-resistant microbial biofilm and theinability of dermal and/or epidermal cells to respond to reparativestimuli.

As explained by Frykberg and Banks (Advances in Wound Care, 2015, Vol.4(9), 560-582), which is incorporated herein by reference in itsentirety, the healing process of wounds is highly complex and isdependent on an intricate interplay between numerous factors working inconcert to restore injured skin towards repaired barrier function.Chronic ulcers are different from acute wounds, which progress in anordered and timely manner through four temporarily and spatiallyoverlapping phases: hemostasis, inflammation, proliferation andremodeling. In contrast to acute wounds, chronic ulcers often stall inthe inflammation phase of healing. Despite differences in etiology atthe molecular level, chronic ulcers share certain common features,including excessive levels of pro-inflammatory cytokines, proteases,reactive oxygen species (ROS), reactive nitrogen species (RNS) andsenescent cells, as well as the existence of persistent infection, and adeficiency of stem cells that are often also dysfunctional. Due torepeated tissue injury, microorganisms and platelet-derived factors,such as transforming growth factor-β (TGF-β) or extracellular matrix(ECM) fragment molecules, stimulate the constant influx of immune cells;the pro-inflammatory cytokine cascade therefore becomes amplified andpersists for a prolonged time, leading to elevated levels of proteases.In chronic ulcers, protease levels exceed those of their respectiveinhibitors, leading to destruction of ECM and degradation of growthfactors and their receptors. The proteolytic destruction of ECM not onlyprevents the ulcer from moving forward into the proliferative phase, butalso attracts more inflammatory cells, thus amplifying the inflammationcycle.

As discussed in Demidova-Rice (Adv. Skin Wound Care, 2012, 25(7):304-314), which is incorporated herein by reference in its entirety,chronic ulcers often feature persistent infections, formation ofdrug-resistant microbial biofilms, and the inability of dermal and/orepidermal cells to respond to reparative stimuli. In aggregate, thesepathophysiologic phenomena result in the failure of these ulcers to healin contrast to acute wounds, which heal within a normal period of time.

Venous ulcers display profound pathological changes that arise secondaryto venous valvular incompetence in the deep and superficial veins. This,in turn, leads to a constant blood backflow resulting in an increase invenous pressure. Pressure-induced changes in blood vessel wallpermeability then lead to leakage of fibrin and other plasma componentsinto the perivascular space. Accumulation of fibrin has direct andnegative effects on wound healing. It down-regulates collagen synthesis,leads to formation of pericapillary fibrin cuffs that create a barrierfor normal vessel function, and traps blood-derived growth factors.Using confocal microscopy, it has been demonstrated that fibrin depositssurrounding dermal veins are patch-like and discontinuous. This findingquestions the barrier role of fibrin cuffs and suggests the presence ofother yet unknown factors contributing to low oxygen tension found invenous ulcers and surrounding tissues. Identification of these factorsmay reveal novel targets for therapeutic interventions and treatment ofvenous ulcers.

Arterial ulcers occur because of arterial insufficiency caused byatherosclerosis or embolism that can lead to narrowing of arterial lumenand ischemia, which prevents timely healing of minor traumatic injuries.Unlike venous ulcers, which generally arise between the knee and theankle, arterial leg wounds may present at any spot distal to arterialperfusion such as a tip of a toe.

Pressure ulcers develop as a result of prolonged unrelieved pressure andshearing force applied to skin and the underlying muscle tissue leadingto a decrease in oxygen tension, ischemia reperfusion injury, and tissuenecrosis. Pressure ulcers are common in patients with compromisedmobility and decreased sensory perception (neuropathies) and areexacerbated in individuals with arterial and venous insufficiencies.

Other abnormalities leading to development of chronic ulcers in diabeticpatients (also called diabetic foot ulcers) include polyneuropathy,often linked to vascular impairment, deficiencies in muscle metabolism,and a number of microvascular pathologies often caused by hyperglycemia.Macroscopic pathologies seen in chronic, particularly diabetic, ulcersoften are linked to cellular phenotypic abnormalities, including lowmitogenic/motogenic potential and inability to respond to environmentalcues.

Although chronic ulcers described in the present disclosure may havedifferent origins, each ulcer is characterized by a chronically inflamedwound bed and a failure to heal. Excessive recruitment of inflammatorycells often triggered by infection and cell extravasation is facilitatedby disproportionate expression of vascular cell adhesion molecule 1 andinterstitial cell adhesion molecule 1 by resident endothelial cellsInflammatory cells accumulated inside the chronic ulcer produce variousROS that damage structural elements of the ECM and cell membranes andlead to premature cell senescence. In addition to these direct negativeeffects, ROS together with pro-inflammatory cytokines induce productionof serine proteinases and matrix metalloproteinases (MMPs) that degradeand inactivate components of the ECM and growth factors necessary fornormal cell function. Inactivation of proteinase inhibitors byproteolytic degradation augments this process. Therefore, although theproduction of growth factors is often increased in chronic compared withacute wounds, their quantity and bio-availability are significantlydecreased.

Since chronic ulcers are portals for local and systemic infection,chronic ulcers can have particularly devastating effects for patients.Poor healing rates of chronic ulcers with conventional therapies arebelieved to be due to the inadequacy of conventional therapies topromote sufficient migration and proliferation of regenerating cells,chemokines, cytokines, nutrients, and growth factors to the site of theulcer. Vasculopathy and infection lead to chronic inflammation at theulcer site, which is associated with an imbalance of growth factors andproteases coupled with reduced proliferation and migration of cells.Increased MMPs at chronic ulcer sites inhibits growth factors, whichleads to decreased migration, attraction and proliferation offibroblasts, keratinocytes and endothelial cells into the ulcer site forhealing.

While protease digestion of ECM components facilitates cell migrationand proliferation and plays a role in the regulation of inflammatoryprocesses, over-expression of proteases and increased proteaseconcentrations are associated with chronic ulcers. MMPs are a largefamily of closely related zinc-finger proteases that digest ECMcomponents including collagens, fibronectin, laminin, and proteoglycans.In normal healing processes, the proteolytic activities of MMPs andneutrophil elastase (NE) are maintained by endogenous proteaseinhibitors, including tissue inhibitors of matrix metalloproteinases(TIMPs), α2-macroglobin, and α1-proteinase inhibitor. Chronic ulcersinvolve elevated concentrations of proteases and increased proteaseexpression relative to acute wounds, and the increased concentrationcontributes to the stalled healing and digestion of the ECM resulting ina stalled inflammatory phase. Related degradation of the structural andadhesion proteins, growth factors, and growth factor receptors alsostall healing.

Diabetes is a major cause of non-traumatic amputations. Chronic skinulcers, particularly diabetic leg and foot ulcers are a major source ofmorbidity in persons with diabetes. Ulceration, infection, gangrene, andamputation are the significant complications of the disease, estimatedto cost many billions of dollars each year (estimated at $50 billion peryear in the United States alone) and affect hundreds of millions ofpeople worldwide. The four most common types of chronic ulcers are:venous ulcers, arterial ulcers, diabetic ulcers, and decubitus(pressure) ulcers. Infections are common in diabetic patients and areoften more severe than infections found in non-diabetic patients.Persons with diabetes have an increased risk of developing an infectionof any kind, resulting in poor quality of life and risk of limbamputation.

Care for chronic ulcers has been reported to cost 2% to 3% of healthcarebudgets in developed countries. While various wound care products havebeen used for treating normally healing wounds and acute ulcers, thereis a demonstrable lack of evidence demonstrating efficacy for a majorityof existing wound care products for treating chronic ulcers.

As discussed in Han and Ceilley (Adv. Ther., 2017, 34:599-610), which isincorporated herein by reference in its entirety, in addition to lack ofefficacy, existing graft materials have disadvantages, including highexpense, difficulty in handling, delicacy or difficulty in obtaininggraft material, poor adhesion to the ulcer bed, require fenestration,require silicone layers as barrier layers, cannot be combined withvacuum assisted closure (VAC) therapy, and fail to sufficiently reduceulcer area, fail to heal all ulcers, fail to promote formation ofsufficient granulation tissue, or fail to reduce ulcer area at anacceptable closure rate. Further, there is a need for products that canprovide a hemostatic effect in chronic ulcers. Promoting blood clottingis particularly important for patients treated with anticoagulants.

For example, one conventional advanced wound care product, EpiFix®(MiMedx®), is an amniotic membrane allograft made of dehydrated humanamnion/chorion membrane tissue (dHACM). The tissue is derived fromdonated human amniotic membranes and, as such, is difficult to handleduring surgery as well as being very expensive. Further, it has beenobserved that amniotic tissue products such as EpiFix® often float onthe blood in the treatment site and lack a hemostatic effect.

Available membranes and products have been found to induce differentcellular inflammatory responses after implantation. In the highlycomplex chronic ulcer environment, it is unpredictable how a given woundcare product will affect physiological processes after implantation. Todate, existing products have demonstrated high failure rates againstchronic ulcers and there is a demonstrable lack of efficacy due to thecomplexity, intractability and unpredictability of the stalled healingprocess in chronic ulcers. Thus, despite the prevalence of chroniculcers, and the availability of graft materials, there exists a need fora method of healing chronic ulcers using a relatively inexpensive,lightweight, easy-to-handle material that can facilitate successfulhealing of the ulcerated area.

U.S. Pat. No. 6,713,085 and European Patent No. 1,709,981 disclose amultilayer sheet of collagen material comprising (i) a barrier layer ofcollagen material having a smooth face and a rough fibrous face oppositesaid smooth face and (ii) a (spongeous) spongeous matrix layer ofcollagen material connected (“adhered”) to said fibrous face, saidspongeous matrix layer of collagen material having an open “sponge-like”texture, and teach that this multilayer sheet of collagen material canbe used as a substitute of autologous graft for free mucosal grafts orsplit thickness skin grafts (see U.S. Pat. No. 6,713,085, C6, lines16-17). That multilayer sheet of collagen material has been marketedunder the trademark Geistlich Mucograft® as a unique collagen matrix forsoft tissue regeneration in the dental field especially for gain ofkeratinized tissue and for recession coverage. That multilayer sheet ofcollagen material has also been used by Ghanaati et al. for augmentationaround dental implants in patients with former head and neck cancer (seeClin. Oral Invest. (2017), 21:1103-1111) and regeneration of facialsurgical wounds to after skin cancer removal (see J. Cell Commun.Signal. (2016) 10:3-15).

Soft tissue regeneration in the dental field and regeneration of oral orfacial surgical wounds after skin cancer removal involves normal healingprocesses of acute wounds: it is pathophysiologically quite differentfrom treatment of chronic ulcers. As taught by the various abovereferences incorporated herein by reference, due to the distinctpathophysiology of chronic ulcers, products that are effectively used totreat acute wounds indeed typically fail to treat chronic ulcers due tothe different physiological phenomena contributing to the chronicity ofchronic ulcers.

SUMMARY OF THE INVENTION

The present disclosure includes methods of treating a subject sufferingfrom chronic ulcers, notably chronic skin ulcers, by increasing liquiduptake capacity, promoting blood clot formation, promoting a hemostaticeffect and accelerating blood coagulation, attracting cells, promotingcell attachment and cell growth of human dermal fibroblasts, humanepidermal keratinocytes, human endothelial cells, and human pluripotentstem cells, binding and preserving the subject's own growth factors,inhibiting matrix metalloproteinases (MMPs) and other collagenases,restoring the tensile strength of skin at the chronic ulcer site to atleast 70% of its original tensile strength, restoring tissue notablyskin at the chronic ulcer site to color, sheen, and tension of thepatient's skin, or a combination thereof at a chronic ulcer site of thesubject.

In some aspects, the subject suffers from venous ulcers, vascularulcers, arterial ulcers, diabetic ulcers, and decubitus (pressure)ulcers, peripheral vascular disease, cellulitis, osteomyelitis, ulcersat surgical sites including donor sites, graft sites, Mohs surgerysites, laser surgery sites, podiatric surgical sites, dehiscence, or acombination thereof. In some aspects, the subject suffers from venousleg ulcers, diabetic foot ulcers, pressure ulcers, or a combinationthereof. In some aspects, the subject suffers from diabetic foot ulcers(DFU). In some aspects, the subject suffers from venous leg ulcers(VLU).

In some aspects, the subject requires ulcer dressing changes, e.g.,daily, every other day, every three days, every five days or every week.

In some aspects, the subject suffers from diabetes, metabolic disorders,thyroid malfunction or dysfunction, and/or an autoimmune disease. Insome aspects, the subject suffers from hyperglycemia, polyneuropathy(e.g. peripheral sensory neuropathy), vasculopathy, infection, fibrincuff, and/or venous hypertension. In some aspects, the subject has beenor is being treated with corticosteroid therapy, is undergoing radiationtherapy, is receiving anti-coagulation therapy, chemotherapy, or usesdrugs, alcohol, tobacco, or other agents that disrupt the normal healingprocess.

In some aspects, the subject treated according to the method of thepresent disclosure is also treated with compression therapy, vacuumassisted closure (VAC), offloading, negative pressure, hyperbaric oxygentherapy, or a combination thereof. Compression therapy may includetherapeutic compression stockings, multilayer compression wraps,wrapping the foot and/or leg with an ACE bandage or dressing, orcompression boot.

In some aspects, the present disclosure provides a method of treating achronic ulcer in a subject in need thereof comprising

i) cleaning to remove bacteria and other pathogens and/or debriding thechronic ulcer until the edges of the ulcer contain viable tissue;

ii) aseptically implanting into the chronic ulcer of the subject amultilayer sheet of collagen material in dry state comprising (i) abarrier layer of collagen material having a smooth face and a roughfibrous face opposite said smooth face and (ii) a spongeous matrix layerof collagen material connected to said rough fibrous face, saidspongeous matrix layer of collagen material having an open sponge-liketexture, such that said rough fibrous face of said barrier layer ofcollagen material to which is connected said spongeous matrix layer ofcollagen material having an open sponge-like texture faces toward and isadjacent to the bed of the chronic ulcer;iii) hydrating the implanted multilayer sheet of collagen material indry state, generally using blood, a sterile isotonic solution, such ase.g. a sterile saline solution, or a combination thereof; andiv) providing a dressing over the implanted, hydrated multilayer sheetof collagen material, thereby restarting stalled cell migration,proliferation and angiogenesis at the chronic ulcer site.

The term “in a dry state” for the multilayer sheet of collagen materialmeans here that the multilayer sheet of collagen material has a watercontent of 5-20% as determined by Karl-Fischer titration according toPh. Eur. 2.5.12A, USP <921>, which is incorporated herein by referencein its entirety.

The term “barrier” in “barrier layer of collagen material” refers to theproperty of the smooth side of inhibiting direct cell migration throughthe collagen material as described in U.S. Pat. No. 5,837,278, which isreferred to in Example 2 of U.S. Pat. No. 6,713,085 and European PatentNo. 1,709,981.

The term “collagen material” here means a collagen-based material whichcomprises 70-100% (w/w) collagen and 0-70% (w/w) elastin. The elastincontent is here measured by desmosine/iodesmosine determinationaccording to a modification of a known method involving hydrolysis andRP-HPLC (see e.g. Guida E. et al. 1990 Development and validation of ahigh performance chromatography method for the determination ofdesmosines in tissues in Journal of Chromatography or Rodriguqe P 2008Quantification of Mouse Lung Elastin During Prenatal Development in TheOpen Respiratory Medicine Journal). To determine thedesmosine/isodesmosine content of dry elastin, the elastin of thecollagen material is subjected to elastin isolation procedures asdescribed by Starcher and Galione in 1976 (Purification and Comparisonof Elastin from Different Animal Species in Analytical Biochemistry).That collagen material is suitably derived from tissues of naturalorigin which contain such proportions of collagen and elastin. Examplesof such tissues include vertebrate, in particular mammalian (e.g.porcine, bovine, equine, ovine, caprine, lapine) peritoneum orpericardium membrane, placenta membrane, small intestine submucosa(SIS), dermis, dura mater, ligaments, tendons, thoracic diaphragm,omentum, fascie of muscles or organs.

In the present specification the shorter term “fibrous face of themultilayer sheet of collagen material” may be used to designate the“rough fibrous face of said barrier layer of collagen material to whichis connected said spongeous matrix layer of collagen material having anopen sponge-like texture”.

As taught in U.S. Pat. No. 6,713,085, European Patent No. 1,709,981(Geistlich, Schlosser and Boyne), notably in Example 3, and U.S. Pat.No. 5,837,278 (Geistlich, Eckmayer and Boyne), the disclosures of whichis incorporated herein by reference in their entireties, as well as thepresent specification:

the barrier layer of collagen material (i) having a smooth face and arough fibrous face opposite said smooth face of the multilayer sheet ofcollagen material, may derived from a natural collagen membrane, notablya mammalian, in particular bovine, porcine or ovine, peritoneum,pericardium, placenta or basal membrane. That collagen of the barrierlayer of collagen material is usually predominantly collagen I, collagenIII or a mixture thereof. One suitable material for that barrier layerof collagen material (i) is the resorbable porcine bilayer membraneGeistlich Bio-Gide® available from Geistlich Pharma AG, Switzerland.

The collagen of the spongeous matrix layer of collagen material (ii)connected to said rough fibrous face that has an open sponge-liketexture of the multilayer sheet of collagen material, may be formed ofcollagen I, II, III., IV or VII or any combination of those collagentypes. The collagen of spongeous matrix layer of collagen material (ii)is usually predominantly formed of collagen I, collagen III or acombination thereof, e.g. about 87% collagen I and 13% collagen III.

The spongeous matrix layer (ii) of collagen material connected to saidrough fibrous face that has an open sponge-like texture of themultilayer sheet of collagen material, is usually obtained by applying acollagen slurry to the rough fibrous face of the barrier layer ofcollagen material (i) and freeze-drying the combined product.

The multilayer sheet of collagen material has a thickness of about0.5-25 mm.

In some aspects, the multilayer sheet of collagen material of thepresent disclosure has properties such that it allows gaseous exchangeat the chronic ulcer site sufficient to promote healing of the chroniculcer, infiltration of white blood cells, enzymes, cytokines, and growthfactors beneficial for restarting stalled healing.

In some aspects, the present disclosure includes a method for promotingautolytic debridement of the chronic ulcer.

In some aspects, the chronic ulcer extends at least through the dermisand has been present for greater than 4 weeks. In some aspects, thechronic ulcer extends at least through the hypodermis and has beenpresent for greater than 6 weeks. In some aspects, the chronic ulcer hasbeen present for greater than 8, 10, 12, 24 or 40 weeks.

In some aspects, the method further comprises applying a secondarydressing or re-dressing the chronic ulcer after step iv) is performed.

In some aspects, the method further comprises applying sterile saline toremove a dressing material from the multilayer sheet of collagenmaterial after step iv) is performed.

In some aspects, the method further comprises changing the dressing overthe implanted multilayer sheet of collagen material every 1 to 7 daysafter step iv) is performed.

In some aspects, the method further comprises removing exudate from thechronic ulcer site every 1 to 7 days after step iv) is performed.

In some aspects, the method further comprises inspecting the chroniculcer every 1 to 7 days, in particular every week, after step iv) andremoving the dressing after a first visible epithelialization isobserved at the chronic ulcer or removing the implanted multilayer sheetof collagen material and repeating steps i) to iv) if one or more ofredness, swelling, hematomas, blistering, inflammation, excess exudate,infection, and necrosis are observed at the chronic ulcer.

In some aspects, the method further comprises performing one or more oftoe-blood pressure readings, pulse volume recordings, transcutaneousoxygen measurements, and skin perfusion pressure measurements.

In some aspects, the method further comprises one or more of promotingneutrophils and monocytes to localize at the chronic ulcer site,promoting formation of a multi-layered cell structure in the ulcer site,promoting conversion of monocytes to macrophages, promoting secretion ofthe patient's own growth factors, promoting tissue proliferation andcell migration, promoting production and cross-linking of collagen atthe chronic ulcer site, promoting growth of endothelial cells, promotingangiogenesis that was stalled at the chronic ulcer site, promotingformation of a vascular network and granulation, promoting oxygenationof the chronic ulcer site, and reducing one or more of purulentdrainage, erythema, pain, warming, tenderness, induration, and bleedingat the chronic ulcer site.

In some aspects, the present disclosure provides a method for increasingliquid uptake capacity in a chronic ulcer of a subject in need thereof,by aseptically implanting into the chronic ulcer of the subject amultilayer sheet of collagen material in dry state comprising (i) abarrier layer of collagen material having a smooth face and a roughfibrous face opposite said smooth face and (ii) a spongeous matrix layerof collagen material connected to said rough fibrous face, saidspongeous matrix layer of collagen material having an open sponge-liketexture, such that said rough fibrous face of the barrier layer to whichis connected that spongeous matrix layer of collagen material having anopen sponge-like texture faces toward and is adjacent to the bed of thechronic ulcer; and hydrating the implanted multilayer sheet of collagenmaterial, thereby increasing liquid uptake capacity in the chroniculcer. In some aspects, exudate drainage and bleeding from the chroniculcer are inhibited, and floating away of the multilayer sheet ofcollagen material out of the bed of the chronic ulcer is prevented, themultilayer sheet of collagen material sticking to the bed of the chroniculcer, probably due to capillary forces and its high pliability andconformability to uneven surfaces.

In some aspects, the present disclosure provides a method for promotinghemostasis in a chronic ulcer of a subject in need thereof, byaseptically implanting a multilayer sheet of collagen material in drystate comprising (i) a barrier layer of collagen material having asmooth face and a rough fibrous face opposite said smooth face and (ii)a spongeous matrix layer of collagen material connected to said roughfibrous face, said spongeous matrix layer of collagen material having anopen sponge-like texture, such that said rough fibrous face of saidbarrier layer of collagen material to which is connected said spongeousmatrix layer of collagen material having an open sponge-like texturefaces toward and is adjacent to the bed of the chronic ulcer, andhydrating the implanted multilayer sheet of collagen material, therebypromoting hemostasis in the chronic ulcer. In some aspects, blood clotformation in a chronic ulcer is accelerated by at least 1.5- to 4-fold,2-fold, 2.5-fold, 3-fold, or 3.5-fold, compared to blood clot formationin a chronic ulcer in the absence of said implanted multilayer sheet ofcollagen material. In some aspects, blood clot formation in a chroniculcer is accelerated by at least 1.5- to 4-fold, 2-fold, 2.5-fold,3-fold, or 3.5-fold, compared to blood clot formation in a chronic ulcerin the absence of said implanted multilayer sheet of collagen materialfor a subject receiving anti-coagulation therapy. In some aspects, thepresent disclosure provides a method for promoting uptake of red andwhite blood cells into the matrix of the multilayer sheet of collagenmaterial of the present disclosure.

In some aspects, the present disclosure provides a method for bindingand preserving a subject's own growth factors in a chronic skin ulcer ofa subject in need thereof, by aseptically implanting into the chroniculcer of the subject a multilayer sheet of collagen material in drystate comprising (i) a barrier layer of collagen material having asmooth face and a rough fibrous face opposite said smooth face and (ii)a spongeous matrix layer of collagen material connected to said roughfibrous face, said spongeous matrix layer of collagen material having anopen sponge-like texture, such that said rough fibrous face of saidbarrier layer of collagen material to which is connected said spongeousmatrix layer of collagen material having an open sponge-like texturefaces toward and is adjacent to the bed of the chronic skin ulcer; andhydrating the implanted multilayer sheet of collagen material, therebypromoting binding of said subject's own growth factors with themultilayer sheet of collagen material and preservation of said subject'sown growth factors and growth factor activity in the chronic skin ulcer,thereby inducing expression of one or more growth factor-responsivegenes in human dermal fibroblasts, human epidermal keratinocytes, humanendothelial cells, and human pluripotent stem cells in the chronic skinulcer of the subject, and promoting cell growth of one or more humancell types in the chronic skin ulcer.

In some aspects, the growth factors are two or more of transforminggrowth factors (TGFs), fibroblast growth factors (FGFs), epidermalgrowth factor (EGF), Insulin-like Growth Factor (IGF-1),Platelet-derived Growth Factors (PDGFs), and vascular endothelial growthfactors (VEGFs).

In some aspects, said one or more human cell types are humanfibroblasts, epidermal keratinocytes, endothelial cells, and pluripotentstem cells.

In some aspects, the present disclosure provides a method for attractingone or more human cell types to a chronic skin ulcer of a subject inneed thereof, comprising

i) aseptically implanting into the chronic skin ulcer of the subject amultilayer sheet of collagen material in a dry state comprising (i) abarrier layer of collagen material having a smooth face and a roughfibrous face opposite said smooth face and (ii) a layer of collagenmaterial connected to said rough fibrous face, said layer of collagenmaterial having an open sponge-like texture, such that said roughfibrous collagen face of said matrix to which is connected the layer ofcollagen material having an open sponge-like texture faces toward and isadjacent to the bed of the chronic skin ulcer, and(ii) hydrating the implanted multilayer sheet of collagen material,thereby attracting one or more human cell types to the chronic skinulcer.

In some aspects, said one or more human cell types are human dermalfibroblasts, epidermal keratinocytes, endothelial cells, and humanpluripotent stem cells.

In some aspects, the present disclosure provides a method for promotingattachment and growth of one or more human cell types in a chronic skinulcer of a subject in need thereof, by (i) aseptically implanting intothe chronic ulcer of the subject a multilayer sheet of collagen materialin dry state comprising (i) a barrier layer of collagen material havinga smooth face and a rough fibrous face opposite said smooth face and(ii) a matrix layer of collagen material connected to said rough fibrousface, said spongeous matrix layer of collagen material having an opensponge-like texture, such that the rough face of the barrier layer ofcollagen material to which is connected that spongeous matrix layer ofcollagen material having an open sponge-like texture faces toward and isadjacent to the bed of the chronic skin ulcer,

(ii) hydrating the implanted multilayer sheet of collagen material indry state,

(iii) promoting attachment and growth of one or more human cell types inthe chronic skin ulcer, and

(iv) promoting proliferation of one or more human cell types in thechronic skin ulcer.

In some aspects, said one or more human cell types are human dermalfibroblasts, epidermal keratinocytes, endothelial cells, and humanpluripotent stem cells.

In some aspects, the present disclosure provides a method for inhibitingone or more MMPs in a chronic skin ulcer of a subject in need thereof,by aseptically implanting into the chronic ulcer of the subject amultilayer sheet of collagen material in a dry state comprising (i) abarrier layer of collagen material having a smooth face and a roughfibrous face opposite said smooth face and (ii) a layer of collagenmaterial connected to said rough fibrous face, said layer of collagenmaterial having an open sponge-like texture, such that said roughfibrous collagen face of said matrix to which is connected the layer ofcollagen material having an open sponge-like texture faces toward and isadjacent to the bed of the chronic skin ulcer and hydrating theimplanted multilayer sheet of collagen material, thereby inhibiting MMPsand other collagenases in the chronic skin ulcer.

In some aspects, said MMPs are MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 orany combination of those MMP types.

In some aspects, the present disclosure provides a method for producinga kit including a blister, a pouch, a multilayer sheet of collagenmaterial of the present disclosure in the blister, and instructions foruse of the multilayer sheet of collagen material of the presentdisclosure, wherein the instructions for use require a user to takespecific actions including a plurality of the following: asepticallytrimming the multilayer sheet of collagen material to the desired sizeand/or shape to form an implant; using a scalpel, shears, scissors,and/or graspers to trim and/or shape the multilayer sheet of collagenmaterial to form an implant; applying the multilayer sheet of collagenmaterial to the chronic ulcer site with the spongeous layer facing thechronic ulcer; directly applying the multilayer sheet of collagenmaterial to the chronic ulcer site in a dry state; storing themultilayer sheet of collagen material at a temperature of between about10 to about 30° C. prior to the implanting step; implanting themultilayer sheet of collagen material in the chronic ulcer and thencompletely hydrating the multilayer sheet of collagen material in situusing blood, sterile saline solution, or a combination thereof tohydrate the multilayer sheet of collagen material; applying a firstdressing that covers the chronic ulcer site having the multilayer sheetof collagen material implanted therein. In some aspects, the kit furthercomprises instructions for use requiring a user to take further specificactions including a plurality of the following: providing a hydrocolloiddressing over the chronic ulcer site having the multilayer sheet ofcollagen material implanted therein; applying a secondary dressing orre-dressing the chronic ulcer site; applying a non-adhesive secondarydressing or re-dressing; applying sterile saline to remove a dressingmaterial from the multilayer sheet of collagen material; changing thedressing over the implanted multilayer sheet of collagen material every1 to 7 days, in particular every week, after implantation; changing thesecondary dressing over the first dressing every 1 to 7 days afterimplantation; removing exudate from the chronic ulcer site every 1 to 7days after implantation; monitoring the size, shape, color,inflammation, and drainage of the edges of the chronic ulcer site every1 to 7 days after implantation; removing the implanted multilayer sheetof collagen material and repeating the implanting step; ensuring thatthe chronic ulcer site is free of acute infection before implanting themultilayer sheet of collagen material; treating infections at or nearthe chronic ulcer site prior to implanting the multilayer sheet ofcollagen material; identifying patients with allergies or sensitivitiesto porcine or collagen materials prior to implanting the multilayersheet of collagen material; and not implanting the multilayer sheet ofcollagen material in patients that have allergies or sensitivities toporcine or collagen materials.

Other features and characteristics of the subject matter of thisdisclosure, as well as the methods of operation, functions of relatedelements of structure and the combination of parts, and economies ofmanufacture, will become more apparent upon consideration of thefollowing description and the appended claims with reference to theaccompanying drawings, all of which form a part of this specification,wherein like reference numerals designate corresponding parts in thevarious figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various exemplary and non-limitingaspects of the subject matter of this disclosure. In the drawings, likereference numbers indicate identical or functionally similar elements.

FIG. 1 is a schematic view of a multilayer sheet of collagen materialaccording to one aspect of the present disclosure implanted in a chroniculcer site and the healing process effected thereby.

FIG. 2 is a schematic view of a multilayer sheet of collagen materialaccording to one aspect of the present disclosure after days ofimplantation in a chronic ulcer site, with microscopy images of cellsinvolved in the healing process (keratinocytes, fibroblasts andendothelial cells)

FIG. 3 contains photographs of DFU chronic ulcers for five differentpatients before and after treatment during different treatment periods(one week for patient No. 4, three weeks for patients Nos. 1 and 5 andfour weeks for patients Nos. 2 and 3) according to one aspect of thepresent disclosure. The % given are the % of ulcer closure after thedifferent treatment periods (ratio of the ulcer surface after treatmentto the ulcer surface before treatment).

FIG. 4 contains photographs of DFU chronic ulcers for three differentpatients before and after treatment during different periods until 100%ulcer closure (five weeks for two patients and six weeks for onepatient) according to one aspect of the present disclosure.

FIG. 5 is a graph showing the liquid uptake capacity by capillarity ofthe multilayer sheet of collagen material according to one aspect of thepresent disclosure.

FIG. 6A shows attachment and proliferation of adult human dermalfibroblasts fluorescently labelled with phalloidin after 3, 7, 10, and17 days in culture, respectively. FIG. 6B shows attachment andproliferation of human epidermal keratinocytes fluorescently labelledwith phalloidin after 3, 7, 10, and 17 days in culture, respectively.FIG. 6C shows a co-culture of human dermal fibroblasts and humanumbilical vein endothelial cells (HUVECs) fluorescently labeled with4′,6-diamidino-2-phenylindole (DAPI) and anti-human CD31 antibody (leftimage) and with DAPI, Phalloidin and anti-human CD31 antibody (rightimage), after 14 days in culture, respectively.

FIG. 7A shows a full thickness skin model of human dermal fibroblastsand human epidermal keratinocytes seeded on a filter membrane. FIG. 7Bshows a full thickness skin model of human dermal fibroblasts and humanepidermal keratinocytes seeded on the multilayer sheet of collagenmaterial according to one aspect of the present disclosure after 14 daysin culture.

FIG. 8A shows the KN Motif and Ankyrin Repeat Domains 4 (KANK4) geneexpression in adult human fibroblasts in response to TGF-b1 with orwithout the multilayer sheet of collagen material. FIG. 8B shows theMMP-1 gene expression in adult human fibroblasts in response to bFGFwith or without the multilayer sheet of collagen material. FIGS. 8C and8D show the EGR3 gene expression response in human umbilical veinendothelial cells in response to VEGF with or without the multilayersheet of collagen material.

FIG. 9A shows the dose-dependent increase in adult human dermalfibroblast (aHDF) proliferation in serum free basal cell culture mediacontaining extracts of the multilayer sheet of collagen material. FIG.9B shows the dose-dependent increase in adult human dermal fibroblast(aHDF) proliferation in cell culture media containing 10% fetal bovineserum (FBS) and extracts of the multilayer sheet of collagen material.

FIG. 10A is a graph showing the effects of extracts of the multilayersheet of collagen material on epidermal keratinocyte trans-wellmigration. FIG. 10B shows the effect of extracts of extracts of themultilayer sheet of collagen material on epidermal keratinocytetrans-well migration. FIG. 10C shows the effect of the multilayer sheetof collagen material (disk) on epidermal keratinocyte trans-wellmigration.

FIG. 11 shows the effect of the multilayer sheet of collagen material ofthe present disclosure on reducing MMP activity.

FIG. 12 shows the effects of the multilayer sheet of collagen materialof the present disclosure on blood clot formation, in the presence orabsence of Enoxaparin, a low molecular weight heparin.

FIG. 13 shows histological staining of the multilayer sheet of collagenmaterial of the present disclosure soaked with blood usingMasson-Goldner stain (scale bar=200 μm).

FIG. 14 shows the Kaplan-Meier plot of time to heal; 90% of patientshealed within 4 weeks of initiating treatment with the multilayer sheetof collagen material of the present disclosure.

FIG. 15A shows the mean percent wound area reduction plotted over the12-week treatment course with the multilayer sheet of collagen materialof the present disclosure. Error bars represent SD. FIG. 15B shows themeasured wound area by patient plotted over the 12-week treatment coursewith the multilayer sheet of collagen material of the presentdisclosure.

FIG. 16 is representative images of the healing time course for the twostudy patients with Wagner 2 wounds and their respective progression toclosure during treatment with the multilayer sheet of collagen materialof the present disclosure (patients 5 and 7; Table 2). The patient inthe top row had the largest wound in the pilot study and healed in 4weeks. The patient in the lower row is representative of the median,also with complete healing in 4 weeks.

FIG. 17 is images of all Wagner 1 wounds at baseline and at closure orat 12-week study conclusion during treatment with the multilayer sheetof collagen material of the present disclosure (subjects 1-4, 6, 8-10;demographic detailed in Table 2). While patient 1 did not fully healover 12 weeks, the ulcer was substantially reduced in size during thetreatment period.

DETAILED DESCRIPTION

While aspects of the subject matter of the present disclosure may beembodied in a variety of forms, the following description andaccompanying figures are merely intended to disclose some of these formsas specific examples of the subject matter encompassed by the presentdisclosure. Accordingly, the subject matter of this disclosure is notintended to be limited to the forms or embodiments so described andillustrated.

The physiological process of normal wound healing is achieved throughfour temporarily and spatially overlapping phases: hemostasis,inflammation, proliferation, and remodeling phases. Immediately afterinjury, hemostasis occurs and is characterized by vasoconstriction andblood clotting, which prevents blood loss and provides the provisionalmatrix for cell migration. Platelets secrete growth factors andcytokines attract fibroblasts, endothelial cells, and immune cells toinitiate the healing process. The subsequent inflammation phase lasts upto 7 days. The predominant cells at work in this phase are phagocyticcells, such as neutrophils and macrophages. Neutrophils release reactiveoxygen species (ROS) and proteases that prevent bacterial contaminationand cleanse the wound of cellular debris. Blood monocytes arrive at thewound site and differentiate into tissue macrophages. The latter notonly remove bacteria and nonviable tissue by phagocytosis, but alsorelease various growth factors and cytokines recruiting fibroblasts,endothelial cells, and keratinocytes to repair the damaged bloodvessels. As the inflammatory phase subsides accompanied by apoptosis ofimmune cells, the proliferation phase begins. This phase is primarilycharacterized by tissue granulation, formation of new blood vessels(angiogenesis), and epithelialization. The last phase occurs once thewound has closed and may last 1-2 years or longer. During this phase,the provisional matrix is remodeled into organized collagen bundles.

As used herein, in the present context, the term “chronic ulcer” refersto a heterogeneous group of ulcer types including, but not limited todiabetic ulcers, including diabetic foot ulcers as well as otherdiabetic ulcers, including chronic ulcers of the legs and hands, venousulcers, including venous leg ulcers, arterial ulcers, decubitus(pressure) ulcers, varicose ulcers, and stasis ulcers. Chronic ulcersfail to proceed through the normal phases of healing in an orderly andtimely manner. Commonly, a chronic ulcer is stalled in the inflammationphase. Commonly, chronic ulcers fail to achieve sufficient healing after4 weeks.

The closure and regeneration of chronic skin ulcers is a differentmedical problem, involving different mechanisms than normal (acute) skinwounds for the multiple reasons discussed in the present disclosure. Forexample, in subjects having venous leg ulcers, venous hypertension,pressure, and infection can contribute to the stalled healing of thevenous leg ulcer. Particularly, compared to a subject with anormally-healing wound, the subject may have increased pressure in thedistal veins of the legs, excessive fibrin deposition around capillarybeds, enlargement of endothelial pores, decreased oxygen permeabilityand tissue hypoxia, trapped growth factors and inflammatory cells in thefibrin cuff, release of proteolytic enzymes, release of reactive oxygenspecies (ROS), dysregulation of various pro-inflammatory cytokines,growth factors and MMPs. For example, in subjects having diabetic footulcers, polyneuropathy, vasculopathy, pressure, and infection cancontribute to the stalled healing of the diabetic foot ulcer.Particularly, compared to a subject with a normally-healing wound, thesubject may have increased formation of glycoproteins, basement membranethickening, reduced endothelial proliferation, decreased vesselpermeability, altered cell migration, high concentrations ofinflammatory cytokines, cellular senescence, increased protease enzymes,degraded growth factors, receptors, matrix and support structures,decreased angiogenesis, and imbalance of MMPs and TIMPs.

Cellular and molecular data from numerous clinical studies suggest thatmost chronic ulcers get “stuck” in a prolonged inflammatory phase thatis due to the presence of both planktonic (free flowing) and biofilmbacteria in the ulcers. The bacteria stimulate production ofpro-inflammatory cytokines like tumor necrosis factor-α (TNF-α) andinterleukin 1 (IL-1), which act as chemotactic factors (chemicalmessengers} to recruit neutrophils, macrophages, and mast cells into theulcers. The inflammatory cells that are drawn into the ulcers secreteproteases (MMPs, neutrophil elastase, and plasmin) and ROS in an attemptto kill bacteria and detach biofilm colonies that are tightly attachedto the ulcer bed. However, because bacterial biofilms are tolerant toROS as well as antibodies and even antiseptics, the biofilms persist andcontinue to stimulate inflammation. This results in chronically elevatedlevels of proteases and ROS that eventually begin to destroy essentialproteins that are necessary for healing, including growth factors, theirreceptors, and ECM proteins. These “off-target” effects of proteases andROS combine to reduce cell proliferation, migration. and generationfunctional scar matrix. The “biological sum” of this prolongedinflammatory state is a distorted molecular and cellular environmentthat prevents healing. In the simplest terms, the molecular and cellularenvironment between acute healing wounds and chronic ulcers is totallydifferent.

Acute wounds, i.e., those that normally and orderly progress through thehealing process, are characterized by relatively low inflammatorycytokines, low proteases, low ROS, intact functional matrix, highmitogenic activity, and mitotically competent cells. In contrast,chronic ulcers are characterized by one or more of relatively highinflammatory cytokines, relatively high proteases, relatively high ROS,degraded and non-functional matrix, low mitogenic activity, andsenescent cells.

MMPs modify the ECM and modulate the chemical messages important incell-to-cell communication. The MMP gene family contains a zinc2+bindingdomain in their active sites and calcium ions to interconnect folds andmaintain structure. Enzymes are divided into subfamilies of secretoryenzymes (collagenases, gelatinases, stromelysins, unclassified), andmembrane-bound type enzymes (MT-MMPS) based upon structuralcharacteristics and the substrates they preferentially bind. Duringnormal healing, keratinocytes, fibroblasts, macrophages and endothelialcells secrete MMPs and express MT-MMPs on their surfaces. In chroniculcers, excessive protease activity from elevated levels of collagenaseand gelatinase interfere with proper granulation tissue formation. MMPssupport healing, morphogenesis, tissue resorption and remodeling, nervegrowth and hair follicle development. In chronic ulcers, the averagelevel of protease activity was found to be approximately 116-fold higherthan in acute wound fluids. Furthermore, as chronic venous ulcers beganto heal, the levels of protease activity decreased. Similar results werereported for fluids or biopsies of chronic pressure ulcers, where levelsof MMP-2, MMP-9, and MMP-4 were 10 to 25 times higher than levels inacute surgical wound fluids. Levels of the TIMPs, which are the naturalinhibitors of MMPs, were found to be decreased in fluids from chronicvenous ulcers compared to acute mastectomy wound fluids. In non-healingchronic pressure ulcers, MMP-8, the neutrophil-derived collagenase, waselevated, indicating that there may be persistent influx of neutrophilsreleasing MMP-8 and elastase, which could contribute to the destructionof ECM proteins and growth factors that are essential for healing.Chronic venous ulcers were found to have 10-fold to 40-fold higherlevels of neutrophil elastase activity and to have degraded a1-antitrypsin. Elevated MMP-2 and MMP-9 levels in chronic venous ulcersalso were observed to coincide with degradation of fibronectin in thewound bed. Fibronectin is an important multi-domain adhesion proteinthat is present in the ECM and granulation tissue and is important inpromoting epithelial cell migration. Proteases in chronic wound fluidswere shown to rapidly degrade exogenously added growth factors, such asTGF-α, epidermal growth factor (EGF), or platelet-derived growth factor(PDGF), using in-vitro laboratory tests. In contrast, exogenously addedgrowth factors were stable when added to acute surgical wound fluids.

Bacterial burden of the ulcer refers to the biofilm, planktonicorganisms, and toxins in the ulcer. Growth factors are degraded in thepresence of significant quantities of bacteria in the ulcer. Proteaseactivity arising from bacterial proteases and MMPs secreted in responseto bacterial antigen or toxins inactivate local growth factors. Thepresence of fibroblasts enhances the degradation suggesting they may bethe source of the MMPs production. All chronic ulcers have a bacterialload, usually consisting of normal flora. Although not an invasiveinfection, colonization may impede healing by creating apro-inflammatory environment with secreted proteases decreasingavailable growth factor effect.

Bacterial virulence, pathogenicity, bacterial load, and toxins inassociation with host defense determine the extent of inhibition createdby colonizing organisms. The term, critical colonization, describes thesituation where there are no systemic signs of colonization, but healingfails to progress along the anticipated trajectory. In the presence ofreplicating organisms, the ulcer may exhibit excessive drainage, pain,odor, bright red fleshy friable granulation tissue, epithelial islandsor epithelial bridging. For adequate wound healing to progress,bacterial balance must be established by decreasing organisms to a leveleasily managed by host defenses.

Bacterial biofilms are known to contribute to numerous chronicinflammatory diseases, and recent evidence suggests that biofilms alsoplay an important role in impairing healing in chronic ulcers. Woundbacteria that grow in clumps embedded in a thick, self-made, protective,slimy barrier of sugars and proteins are called a wound biofilm.Biofilms are defined as complex, dynamic microbial communities made upof microorganisms (bacteria and fungi) that synthesize and secrete aprotective matrix that attaches the biofilm firmly to the wound surface.They consist of a single bacterial or fungal species or, more commonly,may be poly-microbial, that is, they contain multiple diverse speciesthat are continuously changing. A biofilm is a surrounded by anextracellular polymeric matrix (EPM), which attaches to a surface.Recent studies demonstrate that biofilms are becoming a significantcomponent of infections in humans. Both acute and chronic ulcers aresusceptible to the development of biofilms.

Open ulcers provide a perfect environment for opportunistic: organisms,such as bacteria, to reside and reproduce. Analyses of the microflora ofchronic ulcers (such as pressure and diabetic foot ulcers) demonstrate aphenomenon known as chronic ulcer pathogenic biofilms. Typicalmechanisms by which biofilms impede ulcer healing progress involveheightening the level of inflammation; increasing the amount of ROS andproteases in the wound bed; stimulating overly aggressive immuneresponses; producing detrimental exogenous toxins within the ulcerenvironment; and impairing normal chemokine signaling pathways. Aerobicorganisms within biofilms use oxygen and help to create anaerobic nicheswithin the biofilm matrix that support the development of anaerobeswithin the biofilm. Importantly, the presence of biofilms in an ulcermay affect the healing process without visible clinical signs ofinfection.

Biofilms trigger a chronic inflammatory response that results in theaccumulation of neutrophils and macrophages surrounding biofilms. Theneutrophils and macrophages secrete high levels of ROS that affect thebiofilm and the surrounding tissue. Inflammatory cells also secrete highlevels of proteases (MMPs and elastase) that can help to break down theattachments between biofilms and the tissue, dislodging the biofilmsfrom the tissue. However, the ROS and proteases also damage normalsurrounding tissue, proteins, immune cells, and tissue cells, impairinghealing.

Closely linked to the bacterial bioburden in an ulcer is thepro-inflammatory cytokine profile. In general, fluids from acute healingwounds tend to have an early peak of major pro-inflammatory cytokines,TNT-α and IL-1β and their natural inhibitors, P55 and IL-1 receptorantagonist, within the first few days after injury. This corresponds tothe rapid increase in inflammatory cells in acute wounds. The levels ofpro-inflammatory cytokines begin to decrease after 6 to 7 days as theinflammatory stimuli in acute wounds decrease. However, in a study ofchronic leg ulcers, the levels of inflammatory cytokines, IL-1(3, IL-6and TNT-α were significantly higher than in acute healing wounds, and asthe chronic ulcers began to heal, the levels decreased. These findingsindicate that chronic ulcers have persistently elevated levels ofpro-inflammatory cytokines, but in cases where chronic ulcers begin toheal, the molecular environment changes to a less inflammatoryenvironment.

New blood vessel growth is a vital factor in the development of healthygranulation tissue. At least twenty angiogenic factors have beenidentified. Some promote blood vessel growth as their primary functionlike vascular endothelial growth factor (VEGF); while others, appear topromote neo-vascularization as an additional process. In addition toVEGF, angiogenic factors commonly encountered in the healing woundinclude fibroblastic growth factor acidic and basic (FGFa, FGFb),interleukin-8 (IL-8), platelet-derived growth factor BB (PDGF-BB),transforming growth factor-α (TGF-α), transforming growth factor-β(TGF-β), and tumor necrosis factor-α (TNF-α). When angiogenic factorsare produced in excess of inhibitors wound healing occurs. Inpro-inflammatory states some angiogenic factors are produced in excess,which might account for the production of granulomatous tissue ininfected wounds. However, when angiogenic factor production decreases inresponse to decreased production or inhibition granulation tissue doesnot develop. There are at least thirty angiogenic inhibitors. Angiogenicinhibitors-interferon (IFN-α, β and γ), fibronectin fragment, matrixmetalloproteinase inhibitors (TIMPs), plasminogen activator inhibitor,retinoids, and thrombospondin-1 (TSN-1) balance wound angiogensis.Interestingly, TGF-β exerts opposing effects both as an angiogenicstimulator and as an angiogenic inhibitor.

Cellular aging is a term used to describe the phenotypical changes thatoccur in cells that are slow to function secondary to oxidation ofcellular components. Typically, these changes are seen in older cellsthat have encountered oxidative stress over time. The term has also beenused to refer to cells that are obtained from older individuals and nowfunction less aggressively because of the genetic changes that occur inolder individuals secondary to life-time exposures to reactive oxygenspecies. More recently the term has been used to refer to senescentcells that function as though they were older cells or obtained fromolder individual. These macrophages, fibroblasts and keratinocytes foundat the margin of wound beds respond sluggishly to stimulation withappropriate chemotactic agents or growth factors. Whether the oxidativestress occurs cumulative over decades as in elderly patients orgradually over weeks as with a chronic wound, cellular oxidation conferschanges that preclude RNA and protein synthesis. The inability toaggressively respond to stressors with appropriately synthesized protein(enzymes) confers the phenotype of a non-functional or poorlyfunctioning cell.

Characteristically, elderly skin thins, wrinkles, develops increasedfragility, and becomes more susceptible to ulceration. Decreased dermalturnover, slowed toxin clearance, and inadequate skin immune dysfunctionare also noted Inflammatory cells migrate more slowly into ulcer bedsand a generalized decline in cellular function is observed in aged skinof the elderly. On the other hand, cells residing in the base and marginof ulcer beds fail to properly migrate, secrete, and divide when giventhe usual level and type of stimuli. It is unclear if the failure isrelated to an inability to acquire the message at the level of the cellreceptor, a malfunction in the transmission of the information withinthe cell, or a direct blockage at the level of RNA and proteinsynthesis. Alternatively, the abnormality may arise from chemicalinhibitors present at the receptors or within the cell.

Another key concept that emerged from laboratory analysis demonstratesthat the mitogenic activity of chronic ulcer fluids is dramatically lessthan levels in acute wound fluids. Furthermore, when acute wounds andchronic ulcer fluids were combined, the mitotic activity of acute woundfluids was inhibited. These results show that the proteases in chroniculcer fluids degrade growth factors that are normally present in acutewound fluids and without the essential actions of these growth factors,healing will not progress. In chronic ulcers, the capacity of the woundcells to respond to cytokines and growth factors is altered. Researchsuggests that fibroblasts (cells that manufacture collagen and performother essential functions in wound healing) have a diminished responseto growth factors in chronic ulcers. For example, fibroblast culturesestablished from chronic venous leg ulcers proliferated slowly andformed less dense confluent cultures when compared to normal fibroblastcultures established from uninjured dermis. In another study of chronicvenous leg ulcers that were present for more than 3 years, fibroblastsproliferated poorly in response to PDGF added to cell-culture medium andrapidly approached senescence compared to fibroblasts cultured fromvenous ulcers that had been present for less than 3 years.

As used herein, in the present context, the term “surgical wound” refersto a heterogeneous group of wound types including, but not limited towounds at surgical sites including donor sites, graft sites, Mohssurgery sites, laser surgery sites, podiatric surgical sites,post-surgical sites, and dehiscence.

In certain aspects, the present disclosure includes a multilayer sheetof collagen material for use in accordance with methods of the presentdisclosure. As used herein, the term “pliable” means that the materialconforms and adheres naturally to the chronic ulcer site upon hydration.

The multilayer sheet of collagen material comprises (i) a barrier layerof collagen material having a smooth face and a rough fibrous faceopposite said smooth face and (ii) a spongeous matrix layer of collagenmaterial connected to said rough fibrous face, said spongeous matrixlayer having an open sponge-like texture. In some aspects, themultilayer sheet of collagen material may include i) peritoneal porcinecollagen membranes that are purified and treated such that they containnative (not denatured), insoluble collagen in its natural collagenstructure; and ii) collagen fibers that are purified and treated suchthat they have an open fibrous structure. The multilayer sheet ofcollagen material is acellular, virally-inactivated, and has athree-dimensional multi-layer structure, wherein the layers are notlaminated. As used herein, the term “lower layer” refers to thespongeous layer of the multilayer sheet of collagen material that facesthe chronic ulcer and the “upper layer” is the layer of the multilayersheet of collagen material facing away from the chronic ulcer. The upperlayer protects the ulcer and the open healing process. The upper layerhas a smooth texture with appropriate pull out strength properties toallow suturing. The lower layer is a porous spongeous collagen scaffoldthat is highly biocompatible. In certain aspects, the matrix structureincludes interconnected pores about 0.001 to 1000 μm in diameter, e.g.,10 to 400 μm, 20 to 200 μm, 30 to 180 μm, 40 to 150 μm, or any otherdiameter in the disclosed range. In certain aspects, the porosity isabout 80 to about 98% void volume, e.g., 82 to 96%, 84 to 94%, 86 to92%, 88% to 90%, or any other percentage in the disclosed range. Incertain aspects, the thickness ratio between the lower layer and theupper layer is about 10:1 to 2:1, about 8:1 to 3:1, or about 6:1 to 4:1.In certain aspects, the multilayer sheet of collagen material has aliquid uptake capacity such that the weight of the wet multilayer sheetof collagen material is about 6 to about 15 times, or about 7 to about12 times, or about 8 to about 10 times the weight of the multilayersheet of collagen material in dry state, including uptake of blood. Themultilayer sheet of collagen material includes sterile collagen Type I.In certain aspects, the multilayer sheet of collagen material comprisesgreater than 60%, greater than 80% or greater than 85% collagen Type I.In certain aspects, the multilayer sheet of collagen material has beensterilized by γ irradiation or X-ray irradiation. The multilayer sheetof collagen material has low antigenicity and low immunogenicity.

The multilayer sheet of collagen material has properties such that itcan easily be manipulated, cut and shaped in the surgical theater or inprivate practice. In some aspects, the multilayer sheet of collagenmaterial has not been artificially cross-linked or chemically treated,and has a thickness of ranging from about 0.5 mm to about 25 mm, about0.5 to about 10 mm, about 1 mm to about 8 mm, about 2 mm to about 6 mm,about 2.5 mm to about 5 mm, about 3 mm, about 4 mm, or about 5 mm, orany thickness encompassed by any of the preceding ranges. In someaspects, the multilayer sheet of collagen material is circular with adiameter ranging from about 5 mm to about 40 mm, about 10 mm to about 30mm, about 12 mm to about 26 mm, about 12 mm, about 14 mm, about 16 mm,about 18 mm, or about 20 mm, or any diameter encompassed by any of thepreceding ranges. In some aspects, the multilayer sheet of collagenmaterial is rectangular (including square-shaped) with side lengthsranging from about 5 mm to about 100 mm, about 10 mm to about 80 mm,about 15 mm to about 60 mm, about 20 mm to about 50 mm, about 25 mm toabout 40 mm, about 30 mm to about 35 mm, about 15 mm, about 20 mm, about25 mm, about 30 mm, about 40 mm or about 50 mm, or any lengthencompassed by any of the preceding ranges.

In certain aspects, the multilayer sheet of collagen material of thepresent disclosure comprises, consists essentially of, or consists oftreated porcine peritoneal membranes and treated porcine hides producedhaving the basic and novel structure and properties according to thepresent disclosure. As used herein, the phrase “consisting essentiallyof” in the context of the multilayer sheet of collagen material meansthat the material has not been modified by additional artificialcross-linking, has not had any growth factors or other wound- orulcer-treating agents added to it, has not had any antimicrobial agentsadded to it, and/or has not undergone any modification, treatment oradulteration to materially affect the basic and novel characteristics ofthe multilayer sheet of collagen material of the present disclosure.

In certain aspects, the present disclosure includes a method ofpreparing a multilayer sheet of collagen material for use in accordancewith methods of the present disclosure. In certain aspects, a multilayersheet of collagen material can be prepared in accordance with methodsdisclosed in U.S. Pat. No. 6,713,085 (Geistlich, Schloesser, and Boyne)and U.S. Pat. No. 5,837,278 (Geistlich, Eckmayer and Boyne), thedisclosures of which are incorporated herein by reference in theirentireties. In certain aspects, the method of preparing a multilayersheet of collagen material for use in accordance with methods of thepresent disclosure involves producing materials (A) and (B) andcombining the products to form the multilayer sheet of collagen materialin accordance with the present disclosure.

Method of Producing Material (A). In certain aspects, the method ofproducing material (A) includes mechanically freeing porcine peritonealmembranes from flesh and grease to form porcine peritoneal membranematerial that is free of flesh and grease, washing the freed porcineperitoneal membrane material with water to form a washed and freedporcine peritoneal membrane material, treating the washed and freedporcine peritoneal membrane material with a base (e.g., NaOH at aconcentration of about 0.5 to about 10%, about 1 to about 6%, about 2 toabout 4%, or about 2%) for about 6 to about 20 hours, about 8 to about16 hours, about 10 to about 14 hours, or about 12 hours, to formbase-treated washed and freed porcine peritoneal membrane material,washing the base-treated washed and freed porcine peritoneal membranematerial with water, and acidifying through its entire thickness theresulting base-treated washed and freed porcine peritoneal membranematerial with an acid solution, e.g, HCl, at a concentration of about0.1 to about 1%, about 0.2 to about 0.7%, about 0.3 to about 0.5%, orabout 0.32%, to form an acidified porcine peritoneal membrane material.After acidifying through its entire thickness, the method includeswashing the acidified porcine peritoneal membrane material until a pH ofabout 2 to about 5, about 3 to about 4, or about 3.5 is reached. Incertain aspects, after washing the acidified porcine peritoneal membranematerial, the method includes shrinking the washed acidified porcineperitoneal membrane material with saline solution at a concentration ofabout 4 to about 12%, about 5 to about 10%, about 6% to about 8%, orabout 7% saline. In certain aspects, the method includes neutralizingthe shrunk porcine peritoneal membrane material with a neutralizingagent, e.g., NaHCO₃ solution at a concentration of about 0.1 to about5%, about 0.5 to about 3%, or about 1% NaHCO₃ to form a neutralized andshrunk porcine peritoneal membrane material. In certain aspects, themethod includes washing the neutralized and shrunk porcine peritonealmembrane material with water. In certain aspects, the method includesdehydrating the neutralized and shrunk porcine peritoneal membranematerial to form a dehydrated porcine peritoneal membrane material. Incertain aspects, the dehydrating step is performed using acetone. Incertain aspects, the method includes further degreasing the dehydratedporcine peritoneal membrane material. In certain aspects, the furtherdegreasing step is performed using n-hexane. In certain aspects, themethod includes drying the degreased dehydrated porcine peritonealmembrane material to form a dried porcine peritoneal membrane material.In certain aspects, the drying step is performed using ethanol ether.

Method of Producing Material (B). In certain aspects, the method ofproducing material (B) includes grinding porcine hides to form 1 to 20mm pieces of ground porcine hide, removing water from the ground porcinehides to form collagen fibers, defatting the collagen fibers using asolvent, removing the solvent to form solvent-treated defatted collagenfibers, treating the solvent-treated defatted collagen fibers with astrong inorganic base at a pH of above 12 for about 6 to about 24 hoursto form base-treated collagen fibers, treating the base-treated collagenfibers with a strong inorganic acid at a pH of about 0 to about 1 forabout 1 to about 12 hours to form acid-treated collagen fibers, removingacid by rinsing with water to form rinsed acid-treated collagen fibers,homogenizing the rinsed acid-treated collagen fibers in the present of aswelling regulator to form homogenized collagen fibers, drying thehomogenized collagen fibers to form dried collagen fibers, cleaning thedried collagen fibers using one or more organic solvents, evaporatingthe solvents under vacuum to a solvent residue of less than 1% to formcleaned, dried collagen fibers, and mixing the cleaned, dried collagenfibers with water to form a collagen fiber slurry. In certain aspects,the water-soluble solvent is an alcohol or a ketone. In certain aspects,the defatting step is performed using a chlorinated hydrocarbon or anon-chlorinated hydrocarbon. In certain aspects, the chlorinatedhydrocarbon is dichloroethane, methylene chloride, or a combinationthereof. In certain aspects, the non-chlorinated hydrocarbon is hexane,toluene, or a combination thereof. In certain aspects, the swellingregulator is an inorganic salt. In certain aspects, the homogenizedcollagen fibers are dried by freeze-drying. In certain aspects, the oneor more organic solvents are alcohols, ethers, ketones, chlorinatedhydrocarbons, or combinations thereof.

In certain aspects, a method of preparing a multilayer sheet of collagenmaterial of the present disclosure includes pouring the collagen fiberslurry of material (B) onto the dried porcine peritoneal membranematerial (A) to form a combination product, or alternatively pouring theslurry of collagen material (B) into a recipient and laying the fibrousface of membrane (A) on top of the slurry of collagen material, the w/wratio of slurry of collagen material (B) to the membrane (A) in drystate being generally from 25/1 to 1/5, usually from 15/1 to 1/2,allowing (A) and (B) to combine, and freeze-drying the combined product.In certain aspects, the method includes allowing (A) and (B) to combinefor about 10 to about 60 minutes, about 20 to about 40 minutes, or about30 minutes before freeze-drying.

The present disclosure provides a method for implanting the multilayersheet of collagen material into a chronic ulcer. In some aspects, themethod includes cleaning the chronic ulcer site to remove bacteria andother pathogens as well as debridement so that it is free of debris anddevitalized tissue prior to and/or during implantation of the multilayersheet of collagen material. In some aspects, the chronic ulcer isdebrided. In some aspects, the chronic ulcer site is debrided until theedges of the ulcer contain viable tissue. In some aspects, the chroniculcer site is debrided with a surgical sharp spoon or scalpel prior toand/or during implantation of the multilayer sheet of collagen material.In some aspects, the chronic ulcer site is debrided with gauze prior toand/or during implantation of the multilayer sheet of collagen material.In some aspects, the method includes removing exudate from the chroniculcer site prior to and/or during implantation of the multilayer sheetof collagen material. In some aspects, the method includes reducing orinhibiting bleeding at the chronic ulcer site prior to and/or duringimplantation of the multilayer sheet of collagen material. In someaspects, alcohol-containing disinfectant is not used at the chroniculcer site prior to, during or after implantation of the multilayersheet of collagen material. In some aspects, iodine-containingdisinfectant is not used at the chronic ulcer site prior to, during orafter implantation of the multilayer sheet of collagen material.

In some aspects, the method includes providing the multilayer sheet ofcollagen material in a sterile blister. In some aspects the blister isprovided in a pouch. In some aspects, the method includes asepticallytrimming the multilayer sheet of collagen material to the desired sizeand/or shape to form an implant for the chronic ulcer. In some aspects,the method includes using a scalpel, shears, scissors, and/or graspersto trim and/or shape the multilayer sheet of collagen material to forman implant. In some aspects, the method includes applying the multilayersheet of collagen material to the chronic ulcer site with the lowerlayer facing the chronic ulcer. In some aspects, the method includesdirectly applying the multilayer sheet of collagen material to thechronic ulcer site in a dry state. As used herein, “a dry state” meansthat no soaking or rinsing is performed prior to application. In someaspects, the method includes storing the multilayer sheet of collagenmaterial at a temperature of between about 10 to about 30° C., about 12to about 27° C., or about 15 to about 25° C. prior to the implantingstep.

In some aspects, the method includes fixing the multilayer sheet ofcollagen material in the chronic ulcer site. In some aspects, the methodincludes applying an adhesive, e.g., a surgical or organic adhesive. Insome aspects, no adhesive is used. In some aspects, the method includessuturing and in other aspects, the method does not involve suturing themultilayer sheet of collagen material. In some aspects, the methodincludes implanting the multilayer sheet of collagen material in thechronic ulcer and then completely hydrating the multilayer sheet ofcollagen material in situ. In some aspects, the hydrating is performedusing blood, sterile saline solution, or a combination thereof. In someaspects, the method includes applying a first dressing that covers thechronic ulcer site having the multilayer sheet of collagen materialimplanted therein. In some aspects, the method includes providing ahydrocolloid dressing over the chronic ulcer site having the multilayersheet of collagen material implanted therein.

In some aspects, the method includes applying a secondary dressing orre-dressing the chronic ulcer site, e.g., a composite dressing, a gauzedressing, or a film dressing. In some aspects, the secondary dressing orre-dressing is non-adhesive. In some aspects, the method includesapplying sterile saline to remove a dressing material from themultilayer sheet of collagen material. In certain aspects, the methodincludes periodically (e.g. every day, second day, third day, fourth dayor every week) changing the dressing over the implanted multilayer sheetof collagen material until wound closure. The period for wound closureis generally from 1 to 12 weeks after implantation, usually from 1 to 6weeks after implantation and often from 1 to 4 weeks after implantation.In certain aspects, the method includes changing the secondary dressingover the first dressing every day, second day, every fourth day or everyweek after implantation. In certain aspects, the method includesremoving exudate from the chronic ulcer site every day, second day,third day, fourth day or every week after implantation. In certainaspects, the method includes not using a dressing. In certain aspects,the method includes not using a dressing after a first visibleepithelialization is observed. In certain aspects, the method includescontinuing the monitoring and/or re-dressing steps for 3 to 24 weeks, 4to 12 weeks, or 5 to 10 weeks after implantation.

In certain aspects, the method includes monitoring the size, shape,color, inflammation, and drainage of the edges of the chronic ulcer siteevery day, second day, third day, fourth day or every week afterimplantation. In certain aspects, the method includes removing theimplanted multilayer sheet of collagen material and repeating theimplanting step. In certain aspects, if the monitoring step revealsredness, swelling, hematomas, blistering, inflammation, excess exudate,infection, and/or necrosis at the chronic ulcer site, the methodincludes removing the implanted multilayer sheet of collagen materialand repeating the method steps of the present disclosure.

In certain aspects, the method includes ensuring that the chronic ulcersite is free of necrotic tissue or acute infection before implanting themultilayer sheet of collagen material. In certain aspects, the methodincludes treating infections at or near the chronic ulcer site prior toimplanting the multilayer sheet of collagen material using knownanti-infective therapies, e.g., antibiotics. In certain aspects, themethod includes not using antimicrobials in combination with theimplant. In certain aspects, the method includes identifying patientswith allergies or sensitivities to porcine or collagen materials priorto implanting the multilayer sheet of collagen material.

In certain aspects, the chronic ulcer area is from about 1 cm² to about150 cm², about 3 cm² to about 100 cm², about 4 cm² to about 50 cm², orabout 6 cm² to about 12 cm², or any area encompassed by any of thepreceding ranges. Typically, the chronic ulcer area is from 1 to 20 cm².In some aspects, the present disclosure includes a method for implantingmultiple pieces of the multilayer sheet of collagen material of thepresent disclosure into a chronic ulcer, e.g., pieces having differentsizes and/or shapes.

In certain aspects, the present disclosure includes a method step ofinspecting, monitoring, observing, diagnosing, and/or ensuring thatmalignant degeneration and neoplastic lesions are not present at thechronic ulcer site. In certain aspects, the present disclosure includesclassifying the chronic ulcer. For diabetic foot ulcers, the Wagner,University of Texas, and PEDIS classification systems may be used. Forvenous leg ulcers, the Clinical-Etiology-Anatomy-Pathophysiology (CEAP)classification system may be used. For pressure ulcers, the NationalPressure Ulcer Advisory Panel (NPUAP) classification schemes may beused.

In certain aspects, the present disclosure provides a method forreducing formation of glycoproteins in a chronic ulcer, reducingbasement membrane thickening in a chronic ulcer, increasing vesselpermeability in a chronic ulcer, reducing concentration of inflammatorycytokines in a chronic ulcer, reducing cellular senescence in a chroniculcer, decreasing protease enzymes in a chronic ulcer, inhibitingdegradation of growth factors in a chronic ulcer, inhibiting degradationof receptors in a chronic ulcer, inhibiting degradation of matrixstructures in a chronic ulcer, increasing angiogenesis in a chroniculcer, and/or rebalancing MMPs and TIMPs in a chronic ulcer.

In certain aspects, the present disclosure includes obtainingankle-brachial indices (ABI) at baseline for patients. When the ABI isbelow 0.9, the patient should be classified as having impaired arterialperfusion. In certain aspects, the present disclosure includesperforming one or more of toe-blood pressure readings, pulse volumerecordings, transcutaneous oxygen measurements, and skin perfusionpressure measurements.

In certain aspects, the present disclosure includes using the multilayersheet of collagen material of the present disclosure for promotingneutrophils and monocytes to localize at the chronic ulcer site,promoting formation of a multi-layered cell structure in the ulcer site,promoting conversion of monocytes to macrophages, promoting secretion ofthe patient's own growth factors, promoting tissue proliferation andcell migration, promoting production and cross-linking of collagen atthe chronic ulcer site, promoting growth of endothelial cells, promotingangiogenesis that was stalled at the chronic ulcer site, promotingformation of a vascular network and granulation, promoting oxygenationof the chronic ulcer site, and reducing one or more of purulentdrainage, erythema, pain, warming, tenderness, induration, and bleedingat the chronic ulcer site.

In certain aspects, the present disclosure includes implanting amultilayer sheet of collagen material that is free of or essentiallyfree of one or more of glycosaminoglycan, chondroitin, silicone, cells,growth factors, hydrocolloids, hydrogels, alginate-containing compounds,iodine-containing agents, silver-containing agents, hydrogels,antimicrobial agents, cellulose-containing compounds, gel-formingmaterials, extracellular matrix components, equine-derived collagen,bovine-derived collagen, ovine-derived collagen, humectants, denaturedcollagen, gelatinous collagen, chelating agents (e.g.,ethylenediaminetetraacetic acid (EDTA)), glutaraldehyde, amnioticmembrane, chorion membrane, glucosamine, chitin, cotton, polysaccharide,pectin, crosslinking agents, proteases, human skin, latex, human-derivedmaterial, alcohol, cultured skin materials, mesh, foam, fibers,platelet-derived growth factor, nitric oxide, and intestinalsubmucosa-derived collagen. As used herein, “essentially free” meansthat the subject compound has not been added to the multilayer sheet ofcollagen material and any amount of the subject compound that is in themultilayer sheet of collagen material is present in trace amounts as acontaminant.

In certain aspects, the present disclosure includes a kit including oneor more of a blister, a pouch, a multilayer sheet of collagen materialof the present disclosure, instructions for use of the multilayer sheetof collagen material of the present disclosure, wherein the instructionsfor use require a user to take specific actions including one or more ofthe following: aseptically trimming the multilayer sheet of collagenmaterial to the desired size and/or shape to form an implant; using ascalpel, shears, scissors, and/or graspers to trim and/or shape themultilayer sheet of collagen material to form an implant; applying themultilayer sheet of collagen material to the chronic ulcer site with thelower layer facing the chronic ulcer; directly applying the multilayersheet of collagen material to the chronic ulcer site in a dry state;storing the multilayer sheet of collagen material at a temperaturebetween generally about 10 to about 30° C., usually about 12 to about27° C., or preferably about 15 to about 25° C. prior to the implantingstep; applying an adhesive, e.g., a surgical or organic adhesive; notapplying an adhesive; implanting the multilayer sheet of collagenmaterial in the chronic ulcer and then completely hydrating themultilayer sheet of collagen material in situ using blood, sterilesaline solution, or a combination thereof to hydrate the multilayersheet of collagen material; applying a first dressing that covers thechronic ulcer site having the multilayer sheet of collagen materialimplanted therein; providing a hydrocolloid dressing over the chroniculcer site having the multilayer sheet of collagen material implantedtherein; applying a secondary dressing or re-dressing the chronic ulcersite; applying a non-adhesive secondary dressing or re-dressing;applying sterile saline to remove a dressing material from themultilayer sheet of collagen material; changing the dressing over theimplanted multilayer sheet of collagen every 1 to 7 days afterimplantation; changing the secondary dressing over the first dressingevery 1 to 7 days after implantation; removing exudate from the chroniculcer site every day, second day, third day, fourth day or every weekafter implantation; monitoring the size, shape, color, inflammation, anddrainage of the edges of the chronic ulcer site every 1 to 7 days afterimplantation; removing the implanted multilayer sheet of collagenmaterial and repeating the implanting step; ensuring that the chroniculcer site is free of debris and devitalized tissue as well as bacteriaand other pathogens before implanting the multilayer sheet of collagenmaterial; treating infections at or near the chronic ulcer site prior toimplanting the multilayer sheet of collagen material; identifyingpatients with allergies or sensitivities to porcine or collagenmaterials prior to implanting the multilayer sheet of collagen material;and not implanting the multilayer sheet of collagen material in patientsthat have allergies or sensitivities to porcine or collagen materials.

As shown in FIG. 1 , the chronic ulcer site may include an ulceration ofthe hypodermis, dermis and epidermis layers of the subject's skin. Asdiscussed above, a chronic ulcer is stalled in the healing process andoften involves chronic inflammation, an imbalance of growth factors andproteases, as well as reduced proliferation and migration of cells. Themultilayer sheet of collagen material is implanted into the chroniculcer site according to the method of the present disclosure andinactivates matrix metalloproteinases (MMPs) and binds the patient's owngrowth factors. The multilayer sheet of collagen material promotes cellmigration and proliferation in the chronic ulcer site, and promotesangiogenesis and re-epithelialization.

As shown in FIG. 2 , the present disclosure provides a method ofattracting fibroblasts, keratinocytes, endothelial cells, and bloodvessels to the chronic ulcer site. The present disclosure provides amethod of binding and preserving the patient's own growth factors at thechronic ulcer site, while inhibiting activity of MMPs (e.g., MMP1, MMP2,MMP-3, MMP-8, and MMP9) and other collagenases at the chronic ulcersite, and inhibits degradation of growth factors and the collagenmatrix. In some aspects, the present disclosure provides a method ofbinding and preserving the patient's own growth factors at the chroniculcer site, e.g., including but not limited to transforming growthfactors (e.g., TGF-β), basic fibroblast growth factor (bFGF, also knownas FGF2), epidermal growth factor (EGF), Insulin-like Growth Factor(IGF-1), Platelet-derived Growth Factor (PDGF), vascular endothelialgrowth factor (VEGF), and other bioactive growth factors in the chroniculcer site.

As shown in FIG. 3 and FIG. 4 , the present disclosure unexpectedlyprovides a method of successfully closing chronic ulcers including inpatients suffering from diabetic foot ulcers DFU. As shown in FIG. 3 andFIG. 4 , the present disclosure unexpectedly provides a method ofsuccessfully closing chronic ulcers such as DFU within 1 week to about 6weeks of the treatment of the present disclosure. In some aspects, thepresent disclosure provides a method of decreasing the chronic ulcerarea by about 70% to about 100% within 1 week to about 6 weeks of thetreatment of the present disclosure, with an average of about 2 to about3 weeks for diabetic foot ulcers. As shown in FIG. 3 , in one aspect,the present disclosure provides a method of decreasing the chronic ulcerarea from 12 cm² to about 2 cm² within 3 weeks of the treatment of thepresent disclosure. As shown in FIG. 3 , in one aspect, the presentdisclosure provides a method of decreasing the chronic ulcer area from 4cm² to fully closed within 4 weeks of the treatment of the presentdisclosure. As shown in FIG. 3 , in one aspect, the present disclosureprovides a method of decreasing the chronic ulcer area of a diabeticfoot ulcer from 3 cm² to fully closed within 4 weeks of the treatment ofthe present disclosure. As shown in FIG. 3 , in one aspect, the presentdisclosure provides a method of decreasing the chronic ulcer area of adiabetic foot ulcer from 4 cm² to fully closed within 1 week of thetreatment of the present disclosure. As shown in FIG. 3 , in one aspect,the present disclosure provides a method of decreasing the chronic ulcerarea of a diabetic foot ulcer from 5 cm² to about 1 cm² within 3 weeksof the treatment of the present disclosure.

As shown in FIG. 4 , in one aspect, the present disclosure provides amethod of decreasing the chronic ulcer area of a plantar foot ulcer from5 cm² to fully closed within 5 weeks of the treatment of the presentdisclosure. As shown in FIG. 4 , in one aspect, the present disclosureprovides a method of decreasing the chronic ulcer area of a distal footulcer from 15 cm² to fully closed within 6 weeks of the treatment of thepresent disclosure. As shown in FIG. 4 , in one aspect, the presentdisclosure provides a method of decreasing the chronic ulcer area of aplantar foot ulcer from 4 cm² to fully closed within 5 weeks of thetreatment of the present disclosure.

The method of the present disclosure may reduce basement membranethickening in diabetic foot ulcers, may increase vessel permeability indiabetic foot ulcers, may reduce concentration of inflammatory cytokinesin diabetic foot ulcers, may reduce cellular senescence in diabetic footulcers, may decrease protease enzymes in diabetic foot ulcers, mayinhibit degradation of growth factors in diabetic foot ulcers, mayinhibit degradation of receptors in diabetic foot ulcers, may inhibitdegradation of matrix structures in diabetic foot ulcers, may increaseangiogenesis in diabetic foot ulcers, and/or rebalance MMPs and TIMPs indiabetic foot ulcers.

The present disclosure includes methods for promoting cellularattachment and proliferation. The method involves promoting fibroblast,keratinocyte, endothelial, and pluripotent stem cell attachment andproliferation within the material of the present disclosure in a chroniculcer. As evident in comparison to full thickness skin, the upper layermimics the basement membrane, supporting the attachment and growth ofkeratinocytes and suggesting an enhanced re-epithelialization. The lowerlayer beneath the compact layer accommodates fibroblasts and endothelialcells from the surrounding tissue, which may support healing through theproduction of ECM molecules and provision of nutrients to the epitheliallayer. In some aspects, the present method involves a reassemblingprocess without deleterious chemical crosslinking. The presentdisclosure advantageously provides a suitable ECM that supports cellattachment, migration, proliferation, differentiation and angiogenesisin the chronic ulcer.

In some aspects, the present disclosure includes methods for targetingmultiple proteases in the proteolytic cascade and effectively modulatingMMP activity over time particularly in the hyper-proteolytic environmentof chronic ulcers. In some aspects, the present disclosure includesbinding growth factors and preserving their bioactivity over the courseof at least 72 hours in a chronic ulcer sit. In some aspects, thepresent disclosure includes retaining and protecting endogenous growthfactors in the chronic ulcer and restarting arrested healing to proceed.

In some aspects, the present disclosure includes a method for providinga suitable pH environment in the chronic ulcer site. Pathogenic bacteriain the wound bed can contribute to creating an unsuitable healingenvironment. By providing a slightly acidic pH, growth of pathogenicbacteria and excessive breakdown extracellular matrix may be inhibitedor prevented by the method of the present disclosure, and tissueoxygenation may be increased.

For a chronic ulcer such as a venous leg ulcer (VLU) or a diabetic footulcer (DFU), the method of the present disclosure may inhibit depositionof excess fibrin around capillary beds in the chronic ulcer, inhibitenlargement of endothelial pores in the chronic ulcer, increase oxygenpermeability to tissue in the chronic ulcer, inhibit trapping of growthfactors in the fibrin cuff of the chronic ulcer, inhibit trapping ofinflammatory cells in the fibrin cuff of the chronic ulcer, inhibitrelease of reactive oxygen species in the chronic ulcer, and inhibitdysregulation of pro-inflammatory cytokines, growth factors and MMPs inthe chronic ulcer.

Advantages of the present disclosure include avoiding the need toharvest tissue from the patient, avoiding high costs of currentlyavailable graft materials, which are often difficult to handle forsurgeons and are made from human tissue. In addition, the presentdisclosure has the added advantages of not requiring fenestration andnot requiring pre-treatment of the multilayer sheet of collagenmaterial, e.g., no rinsing or pre-hydrating before implantation, therebyreducing implantation/surgical times, reducing risks of contaminationand infection, and increasing ease of use. Further advantages includeeliminating harvest-site pain and complications, shortening surgicaltimes, and promoting natural tissue coloring, sheen, and structuring.

As used herein, the term “about” when used in connection with anumerical value should be interpreted to include any values which arewithin 5% of the recited value. Furthermore, recitation of the termabout and approximately with respect to a range of values should beinterpreted to include both the upper and lower end of the recitedrange.

The following items are included as part of the present disclosure:

1. A method of treating a chronic ulcer in a subject in need thereof,comprising:

-   -   i) cleaning to remove bacteria and other pathogens and/or        debriding the chronic ulcer until the edges of the ulcer contain        viable tissue;    -   ii) aseptically implanting into the chronic ulcer of the subject        a multilayer sheet of collagen material in dry state        comprising (a) a barrier layer of collagen material having a        smooth face and a rough fibrous face opposite said smooth face        and (b) a spongeous matrix layer of collagen material connected        to said rough fibrous face, said spongeous matrix layer of        collagen material having an open sponge-like texture, such that        the rough fibrous face of said barrier layer of collagen        material to which is connected said spongeous matrix layer of        collagen material having an open sponge-like texture, faces        toward and is adjacent to the bed of the chronic skin ulcer;    -   iii) hydrating the implanted multilayer sheet of collagen        material in dry state; and    -   iv) providing a dressing over the implanted, hydrated multilayer        sheet of collagen material, thereby restarting stalled cell        migration, proliferation and angiogenesis at the chronic ulcer        site.        2. The method of item 1, wherein the collagen of said barrier        layer of collagen material is predominantly collagen I, collagen        III or a mixture thereof.        3. The method of item 1, wherein said the collagen of said        spongeous matrix layer of collagen material is predominantly        collagen I, collagen III or a mixture thereof.        4. The method of item 1, wherein multilayer sheet of collagen        material has a thickness of about 0.5-25 mm.        5. The method of item 1, wherein the chronic ulcer extends at        least through the dermis and has been present for greater than 4        weeks.        6. The method of item 1, wherein the chronic ulcer extends at        least through the hypodermis and has been present for greater        than 6, 8, 10, 12, 24, or 40 weeks.        7. The method of item 1, further comprising applying a secondary        dressing or re-dressing the chronic ulcer after step iv) is        performed.        8. The method of item 1, further comprising applying sterile        saline to remove a dressing material from the multilayer sheet        of collagen material after step iv) is performed.        9. The method of item 1, further comprising changing the        dressing over the implanted multilayer sheet of collagen        material every 1 to 7 days after step iv) is performed.        10. The method of item 1, further comprising removing exudate        from the chronic ulcer site every 1 to 7 days after step iv) is        performed.        11. The method of item 1, further comprising inspecting the        chronic ulcer every 1 to 7 days after step iv) and removing the        dressing after a first visible epithelialization is observed at        the chronic ulcer or removing the implanted multilayer sheet of        collagen material and repeating steps i) to iv) if one or more        of redness, swelling, hematomas, blistering, inflammation,        excess exudate, infection, and necrosis are observed at the        chronic ulcer.        12. The method of item 1, further comprising performing one or        more of toe-blood pressure readings, pulse volume recordings,        transcutaneous oxygen measurements, and skin perfusion pressure        measurements.        13. The method of item 1, further comprising one or more of        promoting neutrophils and monocytes to localize at the chronic        ulcer site, promoting formation of a multi-layered cell        structure in the ulcer site, promoting conversion of monocytes        to macrophages, promoting secretion of the patient's own growth        factors, promoting tissue proliferation and cell migration,        promoting production and cross-linking of collagen at the        chronic ulcer site, promoting growth of endothelial cells,        promoting angiogenesis that was stalled at the chronic ulcer        site, promoting formation of a vascular network and granulation,        promoting oxygenation of the chronic ulcer site, and reducing        one or more of purulent drainage, erythema, pain, warming,        tenderness, induration, and bleeding at the chronic ulcer site.        14. A method of increasing liquid uptake capacity in a chronic        ulcer of a subject in need thereof, comprising:    -   i) aseptically implanting into the chronic ulcer of the subject        a multilayer sheet of collagen material in dry state        comprising (a) a barrier layer of collagen material having a        smooth face and a rough fibrous face opposite said smooth face        and (b) a spongeous matrix layer of collagen material connected        to said rough fibrous face, said spongeous matrix layer of        collagen material having an open sponge-like texture, such that        said rough fibrous face of said barrier layer of collagen        material to which is connected said spongeous matrix layer of        collagen material having an open sponge-like texture faces        toward and is adjacent to the bed of the chronic ulcer; and    -   ii) hydrating the multilayer sheet of collagen material in dry        state, thereby increasing liquid uptake capacity in the chronic        ulcer.        15. The method of item 14, further comprising inhibiting exudate        drainage, bleeding from the chronic ulcer, and preventing        floating away of the multilayer sheet of collagen material out        of the bed of the chronic ulcer.        16. A method of promoting hemostasis in a chronic ulcer of a        subject in need thereof, comprising:    -   i) aseptically implanting a multilayer sheet of collagen        material in dry state comprising (a) a barrier layer of collagen        material having a smooth face and a rough fibrous face opposite        said smooth face and (b) a spongeous matrix layer of collagen        material connected to said rough fibrous face, said spongeous        matrix layer of collagen material having an open sponge-like        texture, such that said rough fibrous face of said barrier layer        of collagen material to which is connected said spongeous matrix        layer of collagen material having an open sponge-like texture        faces toward and is adjacent to the bed of the chronic ulcer;        and    -   ii) hydrating the implanted multilayer sheet of collagen        material, thereby promoting hemostasis in the chronic ulcer.        17. The method of item 16, wherein blood clot formation in a        chronic ulcer is accelerated by at least 2-fold compared to        blood clot formation in a chronic ulcer in the absence of said        implanted multilayer sheet of collagen material.        18. A method of binding and preserving a subject's growth own        factors in a chronic skin ulcer of a subject in need thereof,        comprising:    -   i) aseptically implanting into the chronic skin ulcer of the        subject a multilayer sheet of collagen material in dry state        comprising (a) a barrier layer of collagen material having a        smooth face and a rough fibrous face opposite said smooth face        and (b) a spongeous matrix layer of collagen material connected        to said rough fibrous face, said spongeous matrix layer of        collagen material having an open sponge-like texture, such that        said rough fibrous face of said barrier layer of collagen        material to which is connected said spongeous matrix layer of        collagen material having an open sponge-like texture faces        toward and is adjacent to the bed of the chronic ulcer; and    -   ii) hydrating the implanted multilayer sheet of collagen        material, thereby promoting binding of said subject's own growth        factors with the multilayer sheet of collagen material and        preservation of said subject's own growth factors and growth        factor activity in the chronic skin ulcer thereby inducing        expression of one or more growth factor-responsive genes in one        or more human cell types in the chronic skin ulcer of the        subject.        19. The method of item 18, wherein the growth factors are two or        more of transforming growth factors (TGFs), fibroblast growth        factors (FGFs), epidermal growth factor (EGF), Insulin-like        Growth Factor (IGF-1), Platelet-derived Growth Factors (PDGFs),        and vascular endothelial growth factors (VEGFs).        20. The method of item 18, wherein said one or more human cell        types are human fibroblasts, human epidermal keratinocytes,        human endothelial cells and human pluripotent stem cells.        21. A method of attracting one or more human cell types to a        chronic skin ulcer of a subject in need thereof, comprising:    -   i) aseptically implanting into the chronic skin ulcer of the        subject a multilayer sheet of collagen material in dry state        comprising (a) a barrier layer of collagen material having a        smooth face and a rough fibrous face opposite said smooth face        and (b) a spongeous matrix layer of collagen material connected        to said rough fibrous face, said spongeous matrix layer of        collagen material having an open sponge-like texture, such that        said rough fibrous face of said barrier layer of collagen        material to which is connected said spongeous matrix layer of        collagen material having an open sponge-like texture faces        toward and is adjacent to the bed of the chronic skin ulcer; and    -   ii) hydrating the implanted multilayer sheet of collagen        material in dry state, thereby attracting one or more human cell        types to the chronic skin ulcer.        22. The method of item 21, wherein said one or more human cell        types are human fibroblasts, human epidermal keratinocytes,        human endothelial cells and human pluripotent stem cells.        23. A method of promoting attachment and growth of one or more        human cell types in a chronic skin ulcer of a subject in need        thereof, comprising:    -   i) aseptically implanting into the chronic ulcer of the subject        a multilayer sheet of collagen material in dry state        comprising (a) a barrier layer of collagen material having a        smooth face and a rough face opposite said smooth face and (b) a        spongeous matrix layer of collagen material connected to said        fibrous face, said spongeous matrix layer of collagen material        having an open sponge-like texture, such that the rough face of        said barrier layer of collagen material to which is connected        said spongeous matrix layer of collagen material having an open        sponge-like texture faces toward and is adjacent to the bed of        the chronic skin ulcer;    -   ii) hydrating the multilayer sheet of collagen material in dry        state;    -   iii) promoting attachment and growth of one or more human cell        types in the chronic skin ulcer; and    -   iv) promoting proliferation of one or more human cell types in        the chronic skin ulcer.        24. The method of item 23, wherein said one or more human cell        types are human fibroblasts, human epidermal keratinocytes,        human endothelial cells and human pluripotent stem cells.        25. A method of inhibiting one or more MMPs in a chronic skin        ulcer of a subject in need thereof, comprising    -   i) aseptically implanting into the chronic ulcer of the subject        a multilayer sheet of collagen material in dry state        comprising (a) a barrier layer of collagen material having a        smooth face and a rough face opposite said smooth face and (b) a        spongeous matrix layer of collagen material connected to said        fibrous face, said spongeous matrix layer of collagen material        having an open sponge-like texture, such that the rough face of        said barrier layer of collagen material to which is connected        said spongeous matrix layer of collagen material having an open        sponge-like texture faces toward and is adjacent to the bed of        the chronic skin ulcer; and    -   ii) hydrating the implanted multilayer sheet of collagen        material in dry state, thereby inhibiting MMPs and other        collagenases in the chronic skin ulcer.        26. The method of item 25, wherein the MMPs are of MMP-1, MMP-2,        MMP-3, MMP-8, and MMP-9.        27. The method of any one of items 1-26, wherein the subject        suffers from a venous ulcer, a vascular ulcer, an arterial        ulcer, a diabetic ulcer, a decubitus ulcer, a peripheral        vascular disease, cellulitis, osteomyelitis, an ulcer at a        surgical site, dehiscence, or a combination thereof.        28. The method of any one of items 1-26, wherein the subject        suffers from venous leg ulcers, diabetic foot ulcers, pressure        ulcers, or a combination thereof.        29. The method of any one of items 1-26, wherein the subject        suffers from diabetic foot ulcer (DFU) or venous leg ulcer        (VLU).        30. The method of any one of items 1-26, wherein the subject        suffers from diabetes, metabolic disorders, thyroid malfunction        or dysfunction, and/or an autoimmune disease.        31. The method of any one of items 1-26, wherein the subject        suffers from hyperglycemia, neuropathy, vasculopathy, infection,        fibrin cuff, and/or venous hypertension.        32. The method of any one of items 1-26, wherein the subject has        been or is being treated with corticosteroid therapy, is        undergoing radiation therapy, is receiving anti-coagulation        therapy, chemotherapy, or uses drugs, alcohol, tobacco, or other        agents that disrupt a normal ulcer healing process.        33. The method of any one of items 1-32, further comprising        treating the subject with compression therapy, vacuum assisted        closure (VAC), offloading, negative pressure, hyperbaric oxygen        therapy, or a combination thereof.        34. The method of any one of items 1-33, wherein the multilayer        sheet of collagen material in dry state has physical properties        such that it absorbs about 7 to about 12 times its weight of        biological fluids.        35. The method of any one of items 1-34, wherein the multilayer        sheet of collagen material has been gamma-sterilized, or X-ray        sterilized.        36. The method of any one of items 1-35, wherein the multilayer        sheet of collagen material has not been artificially        cross-linked, has not had any growth factors or other        ulcer-treating agents added to it, and/or has not had any        antimicrobial agents added to it.        37. The method of any one of items 1-36, further comprising,        after 4 to 7 days, removing at least a portion of the implanted        multilayer sheet of collagen material and repeating the method        steps.        38. The method of any one of items 1-37, further comprising        providing a pH of about 3 to about 6.7 or about 3.5 to about        6.25, or about 4 to about 5.5, or about 4.1 to about 5.1, or        about 4.15 to about 4.95, or any pH in any of the recited        ranges, in the chronic ulcer site,        39. A method of treatment comprising manufacturing a kit        comprising providing a pouch having a blister, aseptically        sealing a multilayer sheet of collagen material in dry state        comprising (i) a barrier layer of collagen material having a        smooth face and a rough face opposite said smooth face and (ii)        a spongeous matrix layer of collagen material connected to said        fibrous face, said spongeous matrix layer of collagen material        having an open sponge-like texture, in the blister, and        combining the pouch having the blister and multilayer sheet of        collagen material sealed therein with instructions for use of        the multilayer sheet of collagen material of the present        disclosure in a box, wherein the instructions for use require a        user to take specific actions including a plurality of the        following: aseptically trimming the multilayer sheet of collagen        material to the desired size and/or shape to form an implant;        using a scalpel, shears, scissors, and/or graspers to trim        and/or shape the multilayer sheet of collagen material to form        an implant; applying the multilayer sheet of collagen material        to the chronic ulcer site with the lower layer facing the        chronic ulcer; directly applying the multilayer sheet of        collagen material to the chronic ulcer site in a dry state;        storing the multilayer sheet of collagen material at a        temperature of between about 10 to about 30° C. prior to the        implanting step; implanting the multilayer sheet of collagen        material in the chronic ulcer and then completely hydrating the        multilayer sheet of collagen material in situ using blood,        sterile saline solution, or a combination thereof to hydrate the        multilayer sheet of collagen material; and applying a first        dressing that covers the chronic ulcer site having the        multilayer sheet of collagen material implanted therein.        40. The method of item 39, wherein the instructions for use        require a user to take further specific actions including a        plurality of the following: providing a hydrocolloid dressing        over the chronic ulcer site having the multilayer sheet of        collagen material implanted therein; applying a secondary        dressing or re-dressing the chronic ulcer site; applying a        non-adhesive secondary dressing or re-dressing; applying sterile        saline to remove a dressing material from the multilayer sheet        of collagen material; changing the dressing over the implanted        multilayer sheet of collagen material every 1 to 7 days after        implantation; changing the secondary dressing over the first        dressing every 1 to 7 days after implantation; removing exudate        from the chronic ulcer site every 1 to 7 days after        implantation; monitoring the size, shape, color, inflammation,        and drainage of the edges of the chronic ulcer site every 1 to 7        days after implantation; removing the implanted multilayer sheet        of collagen material and repeating the implanting step; ensuring        that the chronic ulcer site is free of acute infection before        implanting the multilayer sheet of collagen material; treating        infections at or near the chronic ulcer site prior to implanting        the multilayer sheet of collagen material; identifying patients        with allergies or sensitivities to porcine or collagen materials        prior to implanting the multilayer sheet of collagen material;        and not implanting the multilayer sheet of collagen material in        patients that have allergies or sensitivities to porcine or        collagen materials.

The following Examples are merely illustrative and are not intended tolimit the scope of the present disclosure in any way.

Example 1: Preparation of the Multilayer Sheet of Collagen Material

Peritoneal membranes from pigs were purified to be completely free fromflesh and grease by mechanical means, washed under running water andtreated with 2% NaOH solution for 12 hours. The membranes were thenwashed under running water and acidified with 0.32% HCl. After thematerial had been acidified through its entire thickness (for about 15minutes) the material was washed with water until a pH of 3.5 wasobtained. The material was then shrunk with 7% saline solution,neutralised with 1% NaHCO₃ solution and washed under running water. Thematerial was then dehydrated with acetone and degreased with n-hexaneand dried using ethanol ether to obtain a purified collagen materialmembrane (A) in dry state having a smooth face acting as a barrier toprevent passage of cells therethrough and a rough fibrous face oppositesaid smooth face. That purified collagen material membrane (A) in drystate had a water content of 5-20% as determined by Karl-Fischertitration according to Ph. Eur. 2.5.12A, USP <921>.

Porcine hides were ground in a meat grinder to pieces of 1 to 20 mm.Water was removed from the ground porcine hides using a water-solublesolvent such as an alcohol or a ketone. The collagen fibers weredefatted using a chlorinated hydrocarbon such as dichloroethane ormethylene chloride, a non-chlorinated hydrocarbon such as hexane ortoluene, or an ether such as TBME (tertbutyl methyl ether). Afterremoving the solvent, the collagen material was treated with a stronginorganic base (e.g., an inorganic base having a pKa of greater than 10)at a pH above 12 for a period of 6 to 24 hours and treated with a stronginorganic acid (e.g., an inorganic acid having a pKa of less than 0) ata pH of 0 to 1 for a period of 1 to 12 hours. The excess acid wasremoved by rinsing with water and the suspension was homogenized to a0.5 to 2% homogenous suspension of collagen material in the presence ofa swelling regulator such as an inorganic salt. The suspension was driedby freeze-drying and the dry collagen material was successively cleanedwith different organic solvents such as alcohols, ethers, ketones andchlorinated hydrocarbons. The solvents were then evaporated under vacuumto a solvent residue of less than 1%. A slurry of collagen material wasprepared by mixing the cleaned dry collagen material obtained above withacidified water to obtain a new slurry of collagen material (B)containing about 1.5% collagen material and about 0.9% sodium chloride.

The slurry of collagen material (B) was poured on the fibrous face ofthe purified collagen material membrane (A) in dry state, oralternatively, the slurry of collagen material (B) was poured in arecipient and the fibrous face of the purified collagen materialmembrane (A) in dry state was laid on top of the slurry of collagenmaterial, the w/w ratio of slurry of collagen material (B) to thepurified collagen material membrane (A) in dry state being generallyfrom 25/1 to 1/5, usually from 15/1 to 1/2. After about 30 minutes, thecombined product was freeze-dried such as to obtain a multilayer sheetof collagen material in dry state comprising (i) a barrier layer ofcollagen material having a smooth face and a fibrous face opposite saidsmooth face (part of the multilayer sheet of collagen material comingfrom the purified collagen membrane (A)) and (ii) a spongeous matrixlayer of collagen material connected to said fibrous face, saidspongeous matrix layer having an open sponge-like texture. Thatmultilayer sheet of collagen material in dry state had a water contentof 5-20% as determined by Karl-Fischer titration according to Ph. Eur.2.5.12A, USP <921>.

The pH of the multilayer sheet of collagen material was determined inwater according to Ph. Eur. 2.2.3, USP <791>, using 0.01 g/ml samplematerial in water. Additionally, the material was incubated in aphosphate buffered saline (PBS) solution for 48 hours to simulatephysiologic conditions. pH measurements were taken of the extractionsolutions following 48 hours incubation. Measurement of pH extractsolutions was utilized to assess the potential impact of multilayersheet of collagen material on the ulcer environment. Following 48 hoursof incubation, the pH of multilayer sheet of collagen material extractin water was 4.11±0.14 and 6.32±0.08 in PBS. PBS control pH was 7.4.

Example 2: Liquid Uptake Experiments

Starting with multilayer sheet of collagen material in dry stateobtained according to Example 1, the lower part of the material wassoaked in phosphate buffered saline (PBS) and the liquid uptake capacityby capillarity was measured until the sample was completely hydrated.The liquid uptake capacity was calculated by dividing the weight of thewet sample by the weight of the dry sample. As shown in FIG. 5 , theliquid uptake capacity of the multilayer sheet of collagen materialaccording to Example 1 was such that the weight of the wet multilayersheet of collagen material was 9.3±0.9 times the weight of themultilayer sheet of collagen material in dry state. These resultsconfirm that the multilayer sheet of collagen material according toExample 1 absorbs a relatively large volume of physiological fluidspresent in the chronic ulcer site and suggest that the multilayer sheetof collagen material is prevented from being flushed away by fluidspresent in excess in the chronic ulcer site.

Example 3: Cell Culture and Cell Attachment and Cell Growth Assays

Adult human dermal fibroblasts (aHDF, ScienCell Research Laboratories)were cultured in complete cell culture medium consisting of Dulbecco'smodified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS)and 1% Penicillin/Streptomycin solution (both GIBCO, Life TechnologiesCorp.). Basal media used in cell treatments were prepared by adding onlythe penicillin/streptomycin component to DMEM and excluding the serumsupplement. Neonatal human epidermal keratinocytes (HEKs) were obtainedfrom LifeTechnologies and cultured in EpiLife medium (LifeTechnologies,Switzerland) supplemented with 1× Human Keratinocyte Growth Supplement(HKGS, LifeTechnologies). HEKs were cultured as per productinstructions.

For co-culture experiments, angiogenesis-tested aHDF and HUVECs (bothCaltag Medsystems, United Kingdom) were cultured and expanded as perproduct instructions for the Vasculogenesis to Angiogenesis (V2a) Kit(Caltag Medsystems, United Kingdom).

aHDFs were seeded onto the upper layer of the multilayer sheet ofcollagen material at a concentration of 50000 cells in 50 μl medium perscaffold. Extra medium was added after 1 h at room temperature. Cultureconditions were maintained at 37° C., 5% CO2, and humid atmosphere, withmedium changed three times per week. At defined time points, medium wasremoved and the scaffolds were washed in PBS, fixed with 10%neutral-buffered formalin (PFA; Sigma-Aldrich), and permeabilized with a10 min incubation in 0.1% Triton X-100/PBS (Sigma-Aldrich). Scaffoldswere washed in PBS at each intermediate step. Cytoskeletal actinstaining was performed with Alexa Fluor® 488 Phalloidin (MolecularProbes) diluted 1:80 in 1% bovine serum albumin/PBS (Sigma-Aldrich).Sections were imaged using a Yokogawa CV1000 Cell Voyager confocalmicroscope. The pictures obtained are represented in FIGS. 6A and 6B.

For co-culture experiments, angiogenesis-tested HUVECs and aHDF (bothCaltag Medsystems) were seeded onto the smooth face of the multilayersheet of collagen material at a concentration of 40,000 cells in 50 μlmedium per scaffold, at a ratio 1:9. Extra medium was added after 1 h atroom temperature. Culture conditions were maintained at 37° C., 5% CO2,and humid atmosphere, with medium changed three times per week, as perproduct instructions (V2a, Caltag Medsystems). After 14 days in culture,medium was removed and the scaffolds were washed in PBS, fixed with 10%PFA (Sigma-Aldrich), and permeabilized with a 10 min incubation in 0.5%Triton X-100/PBS (Sigma-Aldrich). Scaffolds were washed in PBS at eachintermediate step. CD-31 staining was performed with primary mouseantibody (Dako) diluted 1:50 in 1% BSA/PBS for 2 h at RT.Fluorophore-coupled goat anti-mouse antibody (Molecular Probes) wasdiluted 1:1000 in BSA/PBS and incubated for 1 h at RT. Cytoskeletalactin staining was performed in parallel with Alexa Fluor® 488Phalloidin (MolecularProbes) diluted 1:80 in 1% bovine serum albumin/PBS(Sigma-Aldrich). Additionally, the cell nuclei were stained using a1:2000 dilution of DAPI in PBS (MolecularProbes). Samples were imagedusing a Yokogawa CV1000 Cell Voyager confocal microscope. The picturesobtained are represented in FIG. 6C.

For full thickness skin equivalent co-culture experiments, humankeratinocytes and aHDFs (obtained from CELLnTEC AG, Switzerland) wereseeded onto the smooth face of the multilayer sheet of collagenmaterial, according to the manufacturers' instructions (CELLnTEC AG,Switzerland). At the end of culture, samples were fixed with 10% PFA(Sigma-Aldrich) and histological sections were prepared and stained byan independent contract research organization (Morphisto, Germany). Thepictures obtained are represented in FIGS. 7A and 7B.

As shown in FIG. 6A, the multilayer sheet of collagen material preparedaccording to Example 1 was seeded with the adult human dermalfibroblasts and fluorescently labelled with phalloidin after days 3, 7,10, and 17. The staining demonstrates that the adult human dermalfibroblasts attached and proliferated on the multilayer sheet ofcollagen material. As shown in FIG. 6B, the multilayer sheet of collagenmaterial prepared according to Example 1 was seeded with human epidermalkeratinocytes and fluorescently labelled with phalloidin after days 3,7, 10, and 17. The staining demonstrates that the human epidermalkeratinocytes attached and proliferated in the multilayer sheet ofcollagen material.

FIG. 6C shows a co-culture of human dermal fibroblasts and humanumbilical vein endothelial cells (HUVECs) fluorescently labeled with4′,6-diamidino-2-phenylindole (DAPI) and anti-human CD31 antibody (leftimage) and with DAPI, Phalloidin and anti-human CD31 (right image). Themultilayer sheet of collagen material prepared according to Example 1was shown to serve as a suitable collagen scaffold for cell attachmentand growth and allowed a mixture of adult human dermal fibroblasts andhuman umbilical vein endothelial cells to build a network of bloodvessel-forming cells.

As shown in FIG. 7B, the multilayer sheet of collagen material accordingto the present disclosure serves as a suitable matrix for cellattachment and growth as well as a substrate for a full thickness skinequivalent.

Example 4: Growth Factor Response Assays

Human cells were seeded onto washed multilayer sheet of collagenmaterial according to Example 1 that was previously soaked inphysiological solutions containing recombinant human growth factors.Gene expression analysis showed cell responses specific to activeTGF-b1, bFGF, and VEGF. TGF-b1 contributes in the inflammatory phase andis involved with granulation tissue formation, re-epithelialization,matrix formation and remodeling. bFGF accelerates wound healing andfunctions in granulation tissue formation, re-epithelialization, matrixformation and remodeling. VEGF effects on multiple components of thewound healing cascade, including tissue granulation, angiogenesis andepithelization and collagen deposition.

The multilayer sheet of collagen material of the present disclosure wascut in pieces of ˜8×8 mm and incubated 4 h with recombinant human TGF-b1(50 ng/ml), recombinant human bFGF (50 ng/ml), or recombinant human VEGF(100 ng/ml), respectively (all R&D systems, Switzerland), diluted in PBSor cell culture medium. After incubation, membranes were vigorouslywashed four times for 15 minutes with respective solution. aHDF forTGF-b1 and bFGF-treated membranes and HUVECs for VEGF-treated membranes,respectively, were seeded onto the smooth side of the multilayer sheetof collagen material with a concentration of 50,000 cells per sample. Inindicated experiments, cells were seeded directly onto the plate andmembranes were placed on top of the well avoiding contact with the cellsto evaluate incomplete washing of the membrane. After 1 h (VEGF), 24 h(bFGF), and 48 h (TGF-b1), respectively, cells were lysed and RNA wasextracted.

Growth factor target genes were selected for analysis: KANK4(Hs01057354_m1) for TGF-b1, EGR3 (Hs04935588_m1) for VEGF, and MMP-1(Hs00899658_m1) for bFGF, and glyceraldehyde 3-phosphate dehydrogenase(GAPDH) (Hs02758991_g1) as a reference. Total RNA was prepared withTriZol (Life Technologies, Switzerland), and 500 μg were used forreverse transcription (RT) with Aurum Total RNA Mini Kit (Bio-Rad,Switzerland). PCR was performed with the Light Cycler 480 Probes Master(Roche, Switzerland) on a CFX connect PCR System (Bio-Rad). TaqManprobes were purchased from Applied Biosystems. The mRNA levels werecalculated by normalizing to the housekeeping gene GAPDH.

Gene expression of growth factor-responsive genes was induced in adulthuman dermal fibroblasts and human umbilical vein endothelial cellsgrown on washed multilayer sheet of collagen material according toExample 1 previously soaked in physiological solutions containingrecombinant human growth factors. FIG. 8A shows the KN Motif And AnkyrinRepeat Domains 4 (KANK4) response to TGF-b1. FIG. 8B shows the MMP-1response to bFGF. FIGS. 8C and 8D show the EGR3 response to VEGF.Immediate refers to the gene expression response in cells seeded ontothe multilayer sheet of collagen material immediately after washing. 72hours refers to the gene expression in cells seeded onto the multilayersheet of collagen material after washing and storage for 72 hours.

As shown in FIG. 8A, response to TGF-b1 was preserved when washedmultilayer sheet of collagen material according to Example 1 was storedfor 72 h before seeding. As shown in FIG. 8B, response to bFGF waspreserved. As shown in FIGS. 8C and 8D, response to VEGF was preserved.

These findings demonstrate that the multilayer sheet of collagenmaterial according to Example 1 rapidly adsorbs bioactive TGF-b1, bFGF,and VEGF present in physiological solutions and preserves growth factoractivity during storage.

Example 5: Tissue Extracts

To prepare soluble extracts for cell culture experiments, the multilayersheet of collagen material according to Example 1 was minced, yieldingapproximately 1×1 mm pieces of tissue, which were extracted at 10 mg drytissue/ml in basal media appropriate for the cell type to be evaluated.After overnight extraction at 37° C., the tissue residue was removed bycentrifugation at 3220 RCF for 5 minutes at room temperature, and theextract was sterile filtered using a 0.22 mm filter unit.

Example 6: Proliferation Studies on Human Dermal Fibroblasts

Adult human dermal fibroblasts were plated at a density of 2500cells/well on 96-well plates in complete cell culture medium. After 24hours, the medium was aspirated from the wells and replaced with one ofthe following: DMEM lacking serum (negative control), DMEM plus FBS(positive control), or medium containing extracts of the multilayersheet of collagen material of the present disclosure at 0.5, 1, 2, and 5mg/mL, both with and without FBS supplement. Extracts were prepared bymaking serial dilutions of the original 10 mg/mL extract preparedaccording to Example 5 in basal medium, and supplemented with FBS whereindicated. After 72 hours, a CyQuant assay (Molecular Probes CyQuant,Life Technologies C7026) was performed according to the manufacturer'sinstructions to quantify DNA content (n=6 replicates of samples pertreatment) as a measure of cellular proliferation. DNA content wasquantified by use of a fluorescence plate reader (excitation 485nm/emission 528 nm). Direct cell number was quantified by detectingCyQuant-labelled nuclei in IncuCyte FL. Fluorescent nuclei per imagewere counted and the cell number per well was calculated.

Cell were grown in Dulbecco's modified Eagle's medium (DMEM) containing0% FBS (FIG. 9A) or 10% FBS (FIG. 9B), respectively. The multilayersheet of collagen material extract concentration in the respective DMEMformulation is shown on the x-axis. Each value represents the average±standard deviation of 3 batches, each analyzed in 3 independentexperiments with n=6 six wells per experiment. (p<0.001 compared tocontrol and compared to all other concentrations of the multilayer sheetof collagen material; *, p<0.05 compared to control and compared to 0.5mg/ml).

Extracts of the multilayer sheet of collagen material prepared accordingto Example 5 caused a dose-dependent increase in adult human dermalfibroblast (aHDF) proliferation in both basal and complete cell culturemedia (FIGS. 9A and B). The largest proliferative effect was observedfor the 5 mg/ml concentration of the extract, where cell numberincreased significantly. These results establish (i) that componentswill elute from the multilayer sheet of collagen material underphysiological conditions and (ii) that the multilayer sheet of collagenmaterial components cause aHDF to proliferate.

Example 7: Trans-Well Migration Assays

Trans-well migration assays were performed as described in J. TissueEng. Regen, Med. 2008 Dec. 2(8): 491-498

In vitro trans-well migration of HEK toward extracts of the multilayersheet of collagen material of the present disclosure was evaluated usingxCELLigence RTCA DP Real Time Cell Analyzer (Acea Biosciences),according to manufacturer's instructions. When HEK reached 70-80%confluency, they were starved for 3 hours in unsupplemented EpiLifemedium before use in trans-well migration assays. Then, HEK weretrypsinized, neutralized, and centrifuged as per HEK productinstructions, and resuspended in unsupplemented EpiLife medium.

The extracts of the multilayer sheet of collagen material of the presentdisclosure prepared according to Example 5 in unsupplemented EpiLifemedium were further diluted in unsupplemented EpiLife medium toconcentrations of 0.5, 2, and 5 mg/ml. Unsupplemented EpiLife mediumserved as negative control. A positive control of 10% HKGS inunsupplemented EpiLife medium was also included in each assay. Sampleswere loaded to the lower chamber of each well of the BD plate, the platewas assembled and 50 uL of unsupplemented EpiLife medium was added tothe upper chamber of each well. The plate was equilibrated for 1 h at37° C. Then, background measurement was performed. Approximately 30,000cells were added to the upper chamber of each well in 100 uL ofunsupplemented EpiLife medium. The trans-well migration chamber wasincubated at 37° C. for 16 hours. Trans-well migration was recordedthrough determination of cell index every 5 minutes; cell index dataafter 12 hours were recorded. After 16 hours, cells were fixed with 100%ethanol, and stained with DAPI or crystal violet, respectively, toconfirm migrated cells in the lower chamber. Keratinocyte migration wasfurther confirmed using 2 mm diameter disks of the multilayer sheet ofcollagen material of the present disclosure instead of soluble extractof the multilayer sheet of collagen material of the present disclosure.

FIG. 10A shows the effects of extracts of the multilayer sheet ofcollagen material on human epidermal keratinocyte trans-well migration.Each value represents the average ±standard deviation of 2 batches ofthe multilayer sheet of collagen material, each analyzed in 3independent experiments with n=4 six wells per experiment. #, p<0.001compared to basal medium; *, p<0.05 compared to basal medium. Cellindexes were confirmed with crystal violet staining (FIG. 10B).Keratinocyte migration was further confirmed using 2 mm diameter disksof the multilayer sheet of collagen material of the present disclosureinstead of soluble extract of the multilayer sheet of collagen materialof the present disclosure (FIG. 10C). The cell index observed in themultilayer sheet of collagen material disks was significant compared tonegative control conditions and similar to positive control samples.Thus, multilayer sheet of collagen material tissue in the culture mediumwas capable of directing keratinocyte migration in vitro.

Samples of unsupplemented basal medium containing 0.5, 2, and 5 mg/ml ofextracts from multilayer sheet of collagen material, respectively,demonstrated significantly greater human epidermal keratinocyte (HEK)migration compared with basal medium alone. The experimental groupscontaining 2 and 5 mg/ml of the extract of the multilayer sheet ofcollagen material reached or even exceeded the number of cells detectedin positive controls (10% HKGS). These results demonstrated that themultilayer sheet of collagen material in the basal culture medium wascapable of directing significant HEK migration in vitro.

Example 8: MMP Activity Assays

Samples of the multilayer sheet of collagen material of the presentdisclosure were incubated with solutions containing elevated proteaseactivity (EPA). Using fluorometric assays, matrix metalloproteases(MMPs) activity was measured over time. Samples were incubated insolutions containing human recombinant MMP-2, MMP-9, and MMP-1. After 2hours, supernatant extracts were mixed with fluorogenic substrate andprotease activity was measured kinetically. Specifically, multilayersheet of collagen material according to the present disclosure was cutin pieces weighing between 3 and 4 mg. Human recombinant MMPs (R&DSystems) were activated with p-aminophenylmercuric acetate. 100 ngactivated rhMMP (at 2 ng/μl in buffer) were added per mg of multilayersheet of collagen material and incubated at 37° C. with shaking. After 2h of incubation, residual enzymatic activity in the supernatant wasmeasured by adding the fluorogenic substrate Mca K P L G L Dpa-A-R-NH2(R&D Systems) to an aliquot. Relative fluorescence units were read inkinetic mode with a microplate reader (Synergy H1, BioTek). Residual MMPactivity was determined by comparing V max of the multilayer sheet ofcollagen material supernatant relative to V max of an untreated controland expressed as percentage activity.

As shown in FIG. 11 , the multilayer sheet of collagen material of thepresent disclosure (“Test”) is effective at reducing elevated proteaseactivity. A reduction in protease activity may help rebalance thechronic ulcer site by protecting growth factors and new tissue fromMMPs, which in turn can facilitate healing. Each value represents theaverage ±standard deviation of 1 batch of multilayer sheet of collagenmaterial, each analyzed in 2 independent experiments with n=2 six wellsper experiment.

Example 9: Hemostasis Assays

Pulled apart multilayer sheet of collagen material of the presentdisclosure was mixed with native human blood at a ratio of 5 mg of thematerial per ml of blood by inverting a 15 ml polystyrene tube twice,and incubated at 37° C. The tubes were inverted once every minute untilcoagulation was observed and the respective time was recorded in FIG. 12. As a control, the time to clotting of blood in the absence of themultilayer sheet of collagen material of the present disclosure wasobserved and recorded in FIG. 12 . Further, blood was tested in thepresence of 1 IU/ml of the anti-factor Xa compound enoxaparin with orwithout the multilayer sheet of collagen material of the presentdisclosure and the time to clotting was observed and recorded in FIG. 12.

The multilayer sheet of collagen material of the present disclosure werecompletely soaked with native human blood and samples left at roomtemperature. After 60 minutes, the membrane material with coagulatedblood was fixed in 10% neutral-buffered formalin solution (Sigma) andprocessed for sectioning and histological staining. Paraffin-embeddedsections were stained with standard Masson's Trichrome stain as shown inFIG. 13 (scale bar=200 μm). Red and white blood cells were detectedwithin the collagen fibers of the porous part of the multilayer sheet ofcollagen material of the present disclosure.

Example 10: Chronic Ulcer Results for DFU and VLU for the Method Usingthe Multilayer Sheet of Collagen Material of the Invention Compared to aMethod Using EpiFix®

Human patients suffering from chronic ulcers, particularly diabetic footulcers DFU and venous leg ulcers VLU, were treated according to themethod of the present disclosure. The ulcers were photographed andcharacterized before implantation, soon after implantation, and duringthe treatment period for DFU and VLU similarly to what is shown in FIG.3 and FIG. 4 for DFU. The method of the present disclosure successfullyachieved for both DFU and VLU 80-100% closure of chronic ulcers ofvarious sizes and in various patients within 1 to 6 weeks ofimplantation.

Table 1 summarizes wound closure results in DFU or VLU subjects usingthe method of the present disclosure:

Mean Mean time Number ulcer size to closure Study # of subjects (cm²)(weeks) 1 10 (DFU,  3.3   2.7 Wagner grades 1-2) 2 6 (DFU and VLU,2.5-300 In progress Wagner grades 2-3) 3 3 (VLU, 15   3 to 5 Wagnergrades 1-2) 4 1 (DFU,  9   −3   Wagner grade 1) Control (EpiFix ® 32(DFU)  2.6   3.3 as published in Zelen et al., Int. Wound J., 2016Apr.;13(2):272-82)

In appropriate cases, VAC therapy was used with the multilayer sheet ofcollagen material of the present disclosure and it was observed that themultilayer sheet of collagen material of the present disclosure couldsuccessfully be used with VAC therapy.

Further summaries of clinical experiences are provided in Table 2:

Body Mass Duration Patient Weight Height Index pre-existing Wagner No.Sex Age (lb) (ft/in) (BMI) DFU Grade DFU location 1 M 64 223 6′0″ 30.210 1 Plantar distal central metarsal 2 M 81 273 6′4″ 33.2 4 1 Plantardistal lateral metarsal 3 M 64 250  5′11″ 47.2 6 1 Plantar 2nd toe 4 F57 200 5′7″ 30.4 40 1 Dorsal hallux toe 5 M 43 280 6′4″ 34.1 8 2 Plantarmedial Metatarsal 6 M 73 200 6′0″ 27.1 14 1 Plantar medial Metatarsal 7F 63 190 5′4″ 32.6 24 1 Plantar distal central metatarsal 8 M 68 2106′0″ 28.5 4 1 Medial metatarsal 9 F 75 200 5′3″ 35.4 8 1 Plantar midfoot10 M 59 290 5′8″ 42.8 5 1 Plantar midfoot Mean — 64.7 231.6 5′8″ 34.212.3 n.a. — SD — 10.6 38.1 0.4  6.3 11.5 n.a. —

Patients summarized in Table 2 had failed to heal after a minimum of 4weeks of standard wound care regimens such as collagen alginatedressings, negative pressure therapy, and off-loading. Patients had beenpreviously treated with other wound care regimens for an average of 12.3weeks. The protocol for this study was approved by the WesternInstitutional Review Board (20182784), and activities were conducted inconformance with the ethical guidelines of the Declaration of Helsinki.

Patients were instructed to off-load the limb, given a diabeticoffloading boot and returned weekly for wound evaluation and dressingchange. At each weekly visit, the wound area was examined to identifyindicators of complications such as infection or necrosis. The woundswere photographed and measured for area using acetate tracing and 2Danalysis. A new multilayer sheet of collagen material of the presentdisclosure was applied to non-healed wounds at each visit.

Complete healing was defined as 100% re-epithelialisation withoutdrainage or need for dressing. The primary outcome measure was theproportion of patients healed at or before 12 weeks. The treatingclinician evaluated each DFU for closure each week, and wound healingwas further adjudicated by three plastic surgeons based on the acquiredphotographic images. The time to heal and percent ulcer area reductionwere recorded for each patient. Time to heal was evaluated throughKaplan-Meier analysis using the Prism software (Prism 8, GraphPad; SanDiego, Calif.).

Prior to treatment with the multilayer sheet of collagen material of thepresent disclosure, the mean wound size was 3.3 cm², and the meaninitial wound depth was 0.3 cm. Upon application, it was observed in allinstances that the multilayer sheet of collagen material of the presentdisclosure immediately conformed to the wound surface and absorbed woundfluid, blood, and any added saline. A direct apposition between themultilayer sheet of collagen material of the present disclosure and thewound bed was achieved, maintaining the position of the graft in thebed. During follow-up visits, the superficial dressings were easilyremoved without adherence to the underlying newly formed tissue.Inspection of the wounds revealed no signs of infection or necrosis inany patient at any time point. In plantar DFUs, the graft wasconsistently observed to be completely integrated, replaced by tissue,or resorbed at 1 week after each application, while some residual woundmatrix was consistently detectable in DFU sites in the lateral,posterior, and toe locations.

Wound closure was observed in 9 of 10 patients (90%) by the conclusionof the 12-week period with a mean time to closure of 2.7 weeks (FIG. 14). Complete closure was achieved at 1 week in 3 patients, at 3 weeks in3 patients, and at 4 weeks in 3 patients. In summary, nine of 10 DFUshealed within 4 weeks after beginning treatment. Notably, after 1 week,the mean wound area reduction was 71±26%. Further incremental wound areareduction was observed at 4 weeks (98±6%) and 6 weeks (99±2%) (FIGS. 15Aand 15B). At the 12-week study endpoint, the mean wound area reductionfor all 10 patients was 99±2% (FIG. 15A). Both Wagner 2 wounds healedover the study period (FIG. 16 ). Seven out of eight of the Wagner 1wounds healed (FIG. 17 ).

Those early clinical experiences showed that for both DFU and VLU ulcerclosure times and rates were comparable with or better than the reportedclosure rates and times for the significantly more expensive anddifficult-to-handle advanced wound care product EpiFix®. Moreover, themultilayer sheet of collagen material of the present disclosureintegrated with regenerated tissue at the chronic ulcer site withoutcausing excessive inflammation or dehiscence.

Example 11: Clinical Study in Progress Comparing for DFU Treatment theMethod of the Invention Using the Multilayer Sheet of Collagen Materialto a Method Using a Conventional Wound Care Product

A prospective, multi-center, parallel group randomized controlledclinical trial is conducted to compare treatment of patients sufferingfrom diabetic foot ulcers with the multilayer sheet of collagen materialof the present disclosure in comparison to Fibracol® (collagen plusalginate wound care product). Fibracol® has been commonly used as astandard treatment for diabetic foot wounds for over 15 years.

Human patients that are at least 18 years old and have a diabetic footulcer, Wagner grade 1, extending at least through the dermis and presentfor greater than 4 weeks and less than 1-year, wherein the ulcer is aminimum of 1.0 cm² and a maximum of 25 cm² are included in the trial.

The affected DFU site is prepared in accordance with the presentdisclosure and the multilayer sheet of collagen material of the presentdisclosure or Fibracol is implanted into the DFU site. The study designfor the comparative trial is appropriately in line to be able to comparestandard therapy to the inventive therapy at 12 weeks, which is thestandard time to evaluate for complete closure.

The primary endpoint of the study is the percentage of DFU healed at 12weeks. Secondary endpoints include the percentage of index ulcers healedat 6 weeks, the time to heal within 6 and 12 weeks, the percent AreaReduction (PAR) at 6 and 12 weeks, changes in peripheral neuropathy,changes in wound quality of life (per W-QoL), to remove bacteria andother pathogens at 12 weeks, need for measures to control bleeding,product wastage, and cost to ulcer closure. Complete closure is definedas 100% re-epithelialization without drainage.

It is expected that the primary end point and one or more of thesecondary endpoints will be significantly higher/improved using themultilayer sheet of collagen material of the present disclosure comparedto using Fibracol®.

Interim results from the clinical trial have been obtained. It was foundthat in the patients treated with the multilayer sheet of collagenmaterial of the present disclosure, 14/19 healed, 2/19 failed, and 3/19have not yet completed the study treatment period. In the standard ofcare group, 5/16 healed, 9/16 failed, and 2/16 have not yet completedthe study treatment period.

While the subject matter of this disclosure has been described and shownin considerable detail with reference to certain illustrativeembodiments, including various combinations and sub-combinations offeatures, those skilled in the art will readily appreciate otherembodiments and variations and modifications thereof as encompassedwithin the scope of the present disclosure. Moreover, the descriptionsof such embodiments, combinations, and sub-combinations is not intendedto convey that the claimed subject matter requires features orcombinations of features other than those expressly recited in theclaims. Accordingly, the scope of this disclosure is intended to includeall modifications and variations encompassed within the spirit and scopeof the following appended claims.

The invention claimed is:
 1. A method of treating a chronic ulcer ofskin and surrounding tissues in a subject in need thereof, comprising:i) cleaning to remove bacteria and other pathogens and/or debriding thechronic ulcer of skin and surrounding tissues until the edges of theulcer contain viable tissue; ii) aseptically implanting into the chroniculcer of skin and surrounding tissues of the subject a multilayer sheetof collagen material in dry state comprising (a) a barrier layer ofcollagen material having a smooth face and a rough fibrous face oppositesaid smooth face and (b) a spongeous matrix layer of collagen materialconnected to said rough fibrous face, said spongeous matrix layer ofcollagen material having an open sponge-like texture, such that therough fibrous face of said barrier layer of collagen material to whichis connected said spongeous matrix layer of collagen material having anopen sponge-like texture, faces toward and is adjacent to the bed of thechronic ulcer of skin and surrounding tissues; iii) hydrating theimplanted multilayer sheet of collagen material in dry state usingblood, an isotonic solution or a combination thereof; and iv) providinga dressing over the implanted, hydrated multilayer sheet of collagenmaterial, thereby restarting stalled cell migration, proliferation andangiogenesis at the chronic ulcer site.
 2. The method of claim 1,wherein the collagen of said barrier layer of collagen material ispredominantly collagen I, collagen III or a mixture thereof.
 3. Themethod of claim 1, wherein the collagen of said spongeous matrix layerof collagen material is predominantly collagen I, collagen III or amixture thereof.
 4. The method of claim 1, wherein the multilayer sheetof collagen material has a thickness of about 0.5-25 mm.
 5. The methodof claim 1, wherein the chronic ulcer extends at least through thedermis and has been present for greater than 4 weeks.
 6. The method ofclaim 1, wherein the chronic ulcer extends at least through thehypodermis and has been present for greater than 6 weeks.
 7. The methodof claim 1, further comprising applying a secondary dressing orre-dressing the chronic ulcer after step iv) is performed.
 8. The methodof claim 1, further comprising applying sterile saline to remove adressing material from the multilayer sheet of collagen material afterstep iv) is performed.
 9. The method of claim 1, further comprisingchanging the dressing over the implanted multilayer sheet of collagenmaterial every 1 to 7 days after step iv) is performed.
 10. The methodof claim 1, further comprising removing exudate from the chronic ulcersite every 1 to 7 days after step iv) is performed.
 11. The method ofclaim 1, further comprising inspecting the chronic ulcer every 1 to 7days after step iv) and removing the dressing after a first visibleepithelialization is observed at the chronic ulcer or removing theimplanted multilayer sheet of collagen material and repeating steps i)to iv) if one or more of redness, swelling, hematomas, blistering,inflammation, excess exudate, infection, and necrosis are observed atthe chronic ulcer.
 12. The method of claim 1, further comprisingperforming one or more of toe-blood pressure readings, pulse volumerecordings, transcutaneous oxygen measurements, and skin perfusionpressure measurements.
 13. The method of claim 1, further comprising oneor more of promoting neutrophils and monocytes to localize at thechronic ulcer site, promoting formation of a multi-layered cellstructure in the ulcer site, promoting conversion of monocytes tomacrophages, promoting secretion of the patient's own growth factors,promoting tissue proliferation and cell migration, promoting productionand cross-linking of collagen at the chronic ulcer site, promotinggrowth of endothelial cells, promoting angiogenesis that was stalled atthe chronic ulcer site, promoting formation of a vascular network andgranulation, promoting oxygenation of the chronic ulcer site, andreducing one or more of purulent drainage, erythema, pain, warming,tenderness, induration, and bleeding at the chronic ulcer site.
 14. Themethod of claim 1, further comprising attracting one or more human celltypes to the chronic ulcer of skin and surrounding tissues, wherein saidone or more human cell types are human fibroblasts, human epidermalkeratinocytes, human endothelial cells and human pluripotent stem cells.15. The method of claim 1, further comprising promoting attachment andgrowth of one or more human cell types in the chronic ulcer of skin andsurrounding tissues, wherein said one or more human cell types are humanfibroblasts, human epidermal keratinocytes, human endothelial cells andhuman pluripotent stem cells.
 16. The method of claim 1, furthercomprising inhibiting one or more MMPs in the chronic ulcer of skin andsurrounding tissues, wherein the MMPs are a plurality of MMP-1, MMP-2,MMP-3, MMP-8, and MMP-9.
 17. The method of claim 1, wherein the subjectsuffers from diabetic foot ulcer (DFU) or venous leg ulcer (VLU). 18.The method of claim 1, wherein the multilayer sheet of collagen materialin dry state has physical properties such that it absorbs about 7 toabout 12 times its weight of biological fluids.
 19. The method of claim1, wherein the multilayer sheet of collagen material has not beenartificially cross-linked, has not had any growth factors or otherulcer-treating agents added to it, and/or has not had any antimicrobialagents added to it.
 20. The method of claim 1, further comprising, after4 to 7 days, removing at least a portion of the implanted multilayersheet of collagen material and repeating the method steps.
 21. Themethod of claim 1, further comprising providing a pH of or about 3.5 toabout 6.5 in the chronic ulcer site.
 22. A method of regenerating tissueat a chronic tissue ulcer site of a subject in need thereof, comprising:i) aseptically implanting into the chronic tissue ulcer site amultilayer sheet of collagen material in dry state comprising (a) abarrier layer of collagen material having a smooth face and a roughfibrous face opposite said smooth face and (b) a spongeous matrix layerof collagen material connected to said rough fibrous face, saidspongeous matrix layer of collagen material having an open sponge-liketexture, such that said rough fibrous face of said barrier layer ofcollagen material to which is connected said spongeous matrix layer ofcollagen material having an open sponge-like texture faces toward and isadjacent to the chronic tissue ulcer; and ii) hydrating the multilayersheet of collagen material in dry state using blood, an isotonicsolution or a combination thereof.
 23. The method of claim 22, whereinthe collagen of said barrier layer of collagen material is predominantlycollagen I, collagen III or a mixture thereof.
 24. The method of claim22, wherein the collagen of said spongeous matrix layer of collagenmaterial is predominantly collagen I, collagen III or a mixture thereof.25. The method of claim 22, wherein the multilayer sheet of collagenmaterial has a thickness of about 0.5-25 mm.
 26. The method of claim 22,wherein the chronic ulcer extends at least through the dermis and hasbeen present for greater than 4 weeks.
 27. The method of claim 22,wherein the chronic ulcer extends at least through the hypodermis andhas been present for greater than 6 weeks.
 28. A method of binding andpreserving a subject's own growth factors in a chronic tissue ulcer siteof a subject in need thereof, comprising: i) aseptically implanting intothe chronic tissue ulcer site of the subject a multilayer sheet ofcollagen material in dry state comprising (a) a barrier layer ofcollagen material having a smooth face and a rough fibrous face oppositesaid smooth face and (b) a spongeous matrix layer of collagen materialconnected to said rough fibrous face, said spongeous matrix layer ofcollagen material having an open sponge-like texture, such that saidrough fibrous face of said barrier layer of collagen material to whichis connected said spongeous matrix layer of collagen material having anopen sponge-like texture faces toward and is adjacent to the chronictissue ulcer site; and ii) hydrating the implanted multilayer sheet ofcollagen material using blood, an isotonic solution or a combinationthereof, thereby promoting binding of said subject's own growth factorswith the multilayer sheet of collagen material and preservation of saidsubject's own growth factors and growth factor activity in the chronictissue ulcer site thereby inducing expression of one or more growthfactor-responsive genes in one or more human cell types in the chronictissue ulcer site of the subject.
 29. The method of claim 28, whereinthe growth factors are two or more of transforming growth factors(TGFs), fibroblast growth factors (FGFs), epidermal growth factor (EGF),Insulin-like Growth Factor (IGF-1), Platelet-derived Growth Factors(PDGFs), and vascular endothelial growth factors (VEGFs).
 30. The methodof claim 29, wherein said one or more human cell types are humanfibroblasts, human epidermal keratinocytes, human endothelial cells andhuman pluripotent stem cells.